U.S. patent number 6,277,885 [Application Number 09/491,636] was granted by the patent office on 2001-08-21 for acetylenic aryl sulfonamide and phosphinic acid amide hydroxamic acid tace inhibitors.
This patent grant is currently assigned to American Cyanamid Company. Invention is credited to James M. Chen, Jeremy I. Levin.
United States Patent |
6,277,885 |
Levin , et al. |
August 21, 2001 |
Acetylenic aryl sulfonamide and phosphinic acid amide hydroxamic
acid TACE inhibitors
Abstract
Hydroxamic acids having the formula ##STR1## are useful in
treating disease conditions mediated by TNF-.alpha., such as
rheumatoid arthritis, osteoarthritis, sepsis, AIDS, ulcerative
colitis, multiple sclerosis, Crohn's disease and degenerative
cartilage loss.
Inventors: |
Levin; Jeremy I. (New City,
NY), Chen; James M. (Stoddard Court, NJ) |
Assignee: |
American Cyanamid Company
(Madison, NJ)
|
Family
ID: |
26852147 |
Appl.
No.: |
09/491,636 |
Filed: |
January 27, 2000 |
Current U.S.
Class: |
514/575; 514/112;
514/118; 514/521; 558/386; 558/390; 558/397; 562/622 |
Current CPC
Class: |
A61K
31/5375 (20130101); A61K 31/54 (20130101); C07C
311/29 (20130101); C07D 213/68 (20130101); C07D
309/12 (20130101); C07D 405/12 (20130101); C07F
9/36 (20130101); C07C 2601/04 (20170501) |
Current International
Class: |
A61K
31/5375 (20060101); A61K 31/54 (20060101); C07C
311/29 (20060101); C07C 311/00 (20060101); C07D
405/00 (20060101); C07D 405/12 (20060101); C07D
213/00 (20060101); C07D 213/68 (20060101); C07D
309/12 (20060101); C07D 309/00 (20060101); C07F
9/36 (20060101); C07F 9/00 (20060101); A61K
031/185 (); C07C 259/08 () |
Field of
Search: |
;562/622
;558/386,390,397 ;514/575,521,118 |
References Cited
[Referenced By]
U.S. Patent Documents
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Other References
Shire, M.G., Exp. Opin. Ther. Patents 8(5), 531 (1998). .
Grossman, J.M., Woman's Health, 6(6), 627 (1997). .
Isomaki, P.J., Ann. Med., 29, 499 (1997). .
Camussi, G., Drugs, 55(5), 613 (1998). .
Mathison et al., J. Clin Invest., 81 1925, (1988). .
Miethke et al., J. Exp. Med., 175, 91 (1992). .
Piquet, P. F., J. Exp. Med. 166, 1280 (1987). .
Beuther, B., Ann. Rev., Biochem, 57, 505 (1988). .
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Packer, M., Circulation, 92(6), 1379 (1995). .
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.
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|
Primary Examiner: Raymond; Richard L.
Attorney, Agent or Firm: Hogan, Jr.; John W.
Parent Case Text
This application claims the benefit of U.S. Provisional Application
No. 60/155,204, filed Jan. 27, 1999.
Claims
What is claimed is:
1. Hydroxamic acids having the formula: ##STR24##
where the C(.dbd.O)NHOH moiety and the --NR.sub.5 -- moiety are
bonded to adjacent carbons of group A;
wherein A is phenyl, naphthyl, or phenyl fused to a 5 to 7 membered
saturated or unsaturated cycloalkyl ring;
X is SO.sub.2 or --P(O)R.sub.10 ;
Y is phenyl, or naphthyl with the proviso that X and Z may not be
bonded to adjacent atoms of Y;
Z is O, NH, CH, or S;
R.sub.5 is hydrogen or alkyl of 1-6 carbon atoms;
R.sub.6 and R.sub.7 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, --CN, --CCH;
and R.sub.8 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-6
carbon atoms, alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon
atoms, phenyl, or naphthyl;
R.sub.9 is hydrogen, phenyl, naphthyl, alkyl of 1-6 carbon atoms,
or cycloalkyl of 3-6 carbon atoms;
and R.sub.10 is phenyl, naphthyl, alkyl of 1-6 carbon atoms, or
cycloalkyl of 3-6 carbon atoms,;
or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1 wherein the ring atom of A adjacent to the
the --NR.sup.5 -- is carbon and has a substituent other than
hydrogen.
3. A compound of claim 1 wherein both of the carbons of A adjacent
to the --NR.sup.5 -- has a substituent other than hydrogen, and the
carbon of group A para to the --NR.sup.5 -- group has a substituent
other than hydrogen.
4. A compound of claim 1 in which A is a phenyl wherein both of the
carbons of A adjacent to the --NR.sup.5 -- group has a substituent
other than hydrogen, and the carbon of group A para to the
--NR.sup.5 -- group has a substituent other than hydrogen.
5. A compound according to claim 4 wherein Y is a phenyl ring
substituted at the 1- and 4-positions by X and Z, respectively.
6. A compound according to claim 5 wherein X is SO.sub.2.
7. A compound according to claim 5 wherein X is SO.sub.2 and Z is
oxygen.
8. A compound according to claim S wherein X is SO.sub.2, Z is
oxygen, and R.sub.6 and R.sub.7 are hydrogen.
9. A compound according to claim 5 wherein X is SO.sub.2, Z is
oxygen, R.sub.6 and R.sub.7 are hydrogen, and R.sub.8 is --CH.sub.2
OH or methyl.
10. A compound according to claim 1 which is selected from the
group consisting of:
5-Bromo-2-{[4-(4-cyclobutylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-ami
no}-N-hydroxy-3-methyl-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(4-methylamino-but-2-ynyloxy)-benze
nesulfonyl]-amino}-benzamide;
5-Bromo-2-({4-[4-3-dimethylamino-propylamino)-but-2-ynyloxy]-benzenesulfony
l}-methyl-amino)-N-hydroxy-3-methyl-benzamide;
5-Bromo-2-({4-[4-(2-dimethylamino-ethylamino)-but-2-ynyloxy]-benzenesulfony
l}-methyl-amino)-N-hydroxy-3-methyl-benzamide;
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-carb
oxylic acid hydroxyamide;
5-Bromo-N-hydroxy-3-methyl-2-[methyl-(4-prop-2-ynyloxy-benzenesulfonyl)-ami
no]-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-[methyl-(4-pent-2-ynyloxy-benzenesulfonyl)-ami
no]-benzamide;
5-Bromo-2-[(4-hept-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-met
hyl-benzamide;
5-Bromo-2-[(4-hex-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-meth
yl-benzamide;
5-Bromo-N-hydroxy-2-{[4-(4-methoxy-but-2-ynyloxy)-benzenesulfonyl]-methyl-a
mino}-3-methyl-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(3-phenyl-prop-2-ynyloxy)-benzenesu
lfonyl]-amino}-benzamide;
5-Bromo-N-hydroxy-2-({4-[3-(3-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide;
5-Bromo-N-hydroxy-2-({4-[3-(2-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide;
5-Bromo-N-hydroxy-2-({4-[3-(4-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide;
2-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-5-iodo-3-methy
l-benzamide;
2-[Benzyl-(4-but-2-ynyloxy-benzenesulfonyl)-amino]-N-hydroxy-3,5-diemthyl-b
enzamide;
5-Bromo-2-{[4-(4-diethylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino}
-N-hydroxy-3-methyl-benzamide; or
5-Bromo-N-hydroxy-2-{[4-(4-hydroxy-but-2-ynyloxy)-benzenesulfonyl]-methyl-a
mino}-3-methyl-benzamide;
or pharmaceutically acceptable salts thereof.
11. A pharmaceutical composition comprising a compound having the
formula ##STR25##
where the C(.dbd.O)NHOH moiety and the --NR.sub.5 -- moiety are
bonded to adjacent carbons of group A;
wherein A is phenyl, naphthyl, or phenyl fused to a 5 to 7 membered
saturated or unsaturated cycloalkyl ring;
X is SO.sub.2 or --P(O)R.sub.10 ;
Y is phenyl, or naphthyl with the proviso that X and Z may not be
bonded to adjacent atoms of Y;
Z is O, NH, CH.sub.2 or S;
R.sub.5 is hydrogen or alkyl of 1-6 carbon atoms;
R.sub.6 and R.sub.7 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, --CN, --CCH;
and R.sub.8 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-6
carbon atoms, alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon
atoms, phenyl, or naphthyl;
R.sub.9 is hydrogen, phenyl, naphthyl, alkyl of 1-6 carbon atoms,
or cycloalkyl of 3-6 carbon atoms;
and R.sub.10 is phenyl, naphthyl, alkyl of 1-6 carbon atoms, or
cycloalkyl of 3-6 carbon atoms;
or a pharmaceutically acceptable salt thereof.
12. A method of inhibiting pathological changes mediated by
TNF-.alpha. converting enzyme (TACE) in a mammal in need thereof
which comprises providing to said mammal a therapeutically
effective amount of a compound having the formula ##STR26##
where the C(.dbd.O)NHOH moiety and the --NR.sub.5 -- moiety are
bonded to adjacent carbons of group A;
wherein A is phenyl, naphthyl, or phenyl fused to a 5 to 7 membered
saturated or unsaturated cycloalkyl ring;
X is SO.sub.2 or --P(O)R.sub.10 ;
Y is phenyl, or naphthyl with the proviso that X and Z may not be
bonded to adjacent atoms of Y;
Z is O, NH, CH.sub.2 or S;
R.sub.5 is hydrogen or alkyl of 1-6 carbon atoms;
R.sub.6 and R.sub.7 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, --CN, --CCH;
and R.sub.8 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-6
carbon atoms, alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon
atoms, phenyl, or naphthyl;
R.sub.9 is hydrogen, phenyl, naphthyl, alkyl of 1-6 carbon atoms,
or cycloalkyl of 3-6 carbon atoms;
and R.sub.10 is phenyl, naphthyl, alkyl of 1-6 carbon atoms, or
cycloalkyl of 3-6 carbon atoms;
or a pharmaceutically acceptable salt thereof.
13. The method according to claim 12 wherein the condition treated
is rheumatoid arthritis, graft rejection, cachexia, inflammation,
fever, insulin resistance, septic shock, congestive heart failure,
inflammatory disease of the central nervous system, inflammatory
bowel disease or HIV infection.
Description
FIELD OF INVENTION
This invention relates to acetylenic aryl sulfonamide hydroxamic
acids which act as inhibitors of TNF-.alpha. converting enzyme
(TACE). The compounds of the present invention are useful in
disease conditions mediated by TNF-.alpha., such as rheumatoid
arthritis, osteoarthritis, sepsis, AIDS, ulcerative colitis,
multiple sclerosis, Crohn's disease and degenerative cartilage
loss.
BACKGROUND OF THE INVENTION
TNF-.alpha. converting enzyme (TACE) catalyzes the formation of
TNF-.alpha. from membrane bound TNF-.alpha. precursor protein.
TNF-.alpha. is a pro-inflammatory cytokine that is believed to have
a role in rheumatoid arthritis [Shire, M. G.; Muller, G. W. Exp.
Opin. Ther. Patents 1998, 8(5), 531; Grossman, J. M.; Brahn, E. J.
Women's Health 1997, 6(6), 627; Isomaki, P.; Punnonen, J. Ann. Med.
1997, 29, 499; Camussi, G.; Lupia, E. Drugs, 1998, 55(5), 613.]
septic shock [Mathison, et. al. J. Clin. Invest. 1988, 81, 1925;
Miethke, et. al. J. Exp. Med. 1992, 175, 91.], graft rejection
[Piguet, P. F.; Grau, G. E.; et. al. J. Exp. Med. 1987, 166,
1280.], cachexia [Beutler, B.; Cerami, A. Ann. Rev. Biochem. 1988,
57, 505.], anorexia, inflammation [Ksontini, R,; MacKay, S. L. D.;
Moldawer, L. L. Arch. Surg. 1998, 133, 558.], congestive heart
failure [Packer, M. Circulation, 1995, 92(6), 1379; Ferrari, R.;
Bachetti, T.; et. al. Circulation, 1995, 92(6), 1479.],
post-ischaemic reperfusion injury, inflammatory disease of the
central nervous system, inflammatory bowel disease, insulin
resistance [Hotamisligil, G. S.; Shargill, N. S.; Spiegelman, B.
M.; et. al. Science, 1993, 259, 87.] and HIV infection [Peterson,
P. K.; Gekker, G.; et. al. J. Clin. Invest. 1992, 89, 574;
Pallares-Trujillo, J.; Lopez-Soriano, F. J. Argiles, J. M. Med.
Res. Reviews, 1995, 15(6), 533.]], in addition to its
well-documented antitumor properties [Old, L. Science, 1985, 230,
630.]. For example, research with anti-TNF-.alpha. antibodies and
transgenic animals has demonstrated that blocking the formation of
TNF-.alpha. inhibits the progression of arthritis [Rankin, E. C.;
Choy, E. H.; Kassimos, D.; Kingsley, G. H.; Sopwith, A. M.;
Isenberg, D. A.; Panayi, G. S. Br. J. Rheumatol. 1995, 34, 334;
Pharmaprojects, 1996, Therapeutic Updates 17 (Oct.), aul97-M2Z.].
This observation has recently been extended to humans as well as
described in "TNF-.alpha. in Human Diseases", Current
Pharmaceutical Design, 1996, 2, 662.
It is expected that small molecule inhibitors of TACE would have
the potential for treating a variety of disease states. Although a
variety of TACE inhibitors are known, many of these molecules are
peptidic and peptide-like which suffer from bioavailability and
pharmacokinetic problems. In addition, many of these molecules are
non-selective, being potent inhibitors of matrix metalloproteinases
and, in particular, MMP-1. Inhibition of MMP-1 (collagenase 1) has
been postulated to cause joint pain in clinical trials of MMP
inhibitors [Scrip, 1998, 2349, 20] Long acting, selective, orally
bioavailable non-peptide inhibitors of TACE would thus be highly
desirable for the treatment of the disease states discussed
above.
Examples of sulfonamide hydroxamic acid MMP/TACE inhibitors in
which a 2 carbon chain separates the hydroxamic acid and the
sulfonamide nitrogen, as shown below, are disclosed in WIPO
international publications WO9816503, WO9816506, WO9816514 and
WO9816520 and U.S. Pat. No. 5,776,961. ##STR2##
U.S. Pat. Nos. 5,455,258, 5,506,242, 5,552,419, 5,770,624,
5,804,593 and 5,817,822 as well as European patent application
EP606,046A1 and WIPO international publications WO9600214 and
WO9722587 disclose non-peptide inhibitors of matrix
metalloproteinases and/or TACE of which the aryl sulfonamide
hydroxamic acid shown below, in which 1 carbon separates the
hydroxarnic acid and the sulfonamide nitrogen, is representative.
Additional publications disclosing sulfonamide based MMP inhibitors
which are variants of the sulfonamidehydroxamate shown below, or
the analogous sulfonamide-carboxylates, are European patent
applications EP-757037-A1 and EP-757984-A1 and WIPO international
publications WO9535275, WO9535276, WO9627583, WO9719068, WO9727174,
WO9745402, WO9807697, and WO9831664, WO9833768, WO9839313,
WO9839329, WO9842659 and WO9843963. The discovery of this type of
MMP inhibitor is further detailed by MacPherson, et. al. in J. Med.
Chem., (1997), 40, 2525 and Tamura, et. al. in J. Med. Chem.
(1998), 41, 640. ##STR3##
Publications disclosing .beta.-sulfonamide-hydroxamate inhibitors
of MMPs and/or TACE in which the carbon alpha to the hydroxamic
acid has been joined in a ring to the sulfonamide nitrogen, as
shown below, include U.S. Pat. No. 5,753,653, WIPO international
publications WO9633172, WO9720824, WO9827069, WO9808815, WO9808822,
WO9808823, WO9808825, WO9834918, WO9808827, Levin, et. al. Bioorg.
& Med. Chem. Letters 1998, 8, 2657 and Pikul, et. al. J. Med.
Chem. 1998, 41, 3568. ##STR4##
The patent applications DE19,542,189-A1, WO9718194, and EP803505
disclose additional examples of cylic sulfonamides as MMP and/or
TACE inhibitors. In this case the sulfonamide-containing ring is
fused to a aromatic or heteroaromatic ring. ##STR5##
Analogous to the sulfonamides are the phosphinic acid amide
hydroxamic acid MMP/TACE inhibitors, exemplified by the structure
below, which have been disclosed in WIPO international publication
WO9808853. ##STR6##
Sulfonamide MMP/TACE inhibitors in which a thiol is the zinc
chelating group, as shown below, have been disclosed in WIPO
international application 9803166. ##STR7##
It is an object of this invention to disclose aryl sulfonamide
hydroxamic acid MMP/TACE inhibitors in which the sulfonyl aryl
group is para-substituted with a substituted butynyl moiety or a
propargylic ether, amine or sulfide. These compounds provide
enhanced levels of inhibition of the activity of TACE in vitro and
in a cellular assay and/or selectivty over MMP-1. These compounds
may therefore be used in the treatment of diseases mediated by
TNF.
DETAILED DESCRIPTION OF THE INVENTION
The TACE and MMP inhibiting ortho-sulfonamido aryl hydroxamic acids
of the present invention are represented by the formula:
##STR8##
where the C(.dbd.O)NHOH moiety and the --NR.sup.5 --moiety are
bonded to adjacent carbons of group A;
Wherein A is phenyl, naphthyl, or phenyl fused to a 5 to 7 membered
saturated or unsaturated cycloalkyl ring, a 5 to 9 membered
saturated or unsaturated heterocycloalkyl ring having 1 or 2
heteroatoms selected from N, NR9, O or S, or a heteroaryl ring
having 5-10 members and from 1-3 heteroatoms selected from N, NR9,
O or S;
X is SO.sub.2 or --P(O)R.sub.10 ;
Y is phenyl, naphthyl or 5-10 membered heteroaryl having from 1 to
3 heteroatoms selected from N, NR.sub.9, O or S; with the proviso
that X and Z may not be bonded to adjacent atoms of Y;
Z is O, NH, CH.sub.2, or S;
R.sub.5 is hydrogen or alkyl of 1-6 carbon atoms;
or R.sub.5 --N--A--, can form a benzazepine, benzoxazepine,
benzothiazepine, benzodiazepine, benzazocine, benzodiazocine,
benzoxazocine or benzothiazocane ring which may be optionally fused
to another benzene ring;
R.sub.6 and R.sub.7 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, --CN, --CCH;
and R.sub.8 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-6
carbon atoms, alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon
atoms, phenyl, naphthyl, 5 to 10 membered heteroaryl having from 1
to 3 heteoatoms selected from N, NR9, O or S, or 5 to 9 membered
heterocycloalkyl having 1 or 2 heteroatoms selected from N, NR9, O
or S;
R.sub.9 is hydrogen, phenyl, naphthyl, alkyl of 1-6 carbon atoms,
or cycloalkyl of 3-6 carbon atoms;
and R.sub.10 is phenyl, naphthyl, alkyl of 1-6 carbon atoms,
cycloalkyl of 3-6 carbon atoms, 5 to 10 membered heteroaryl having
from 1 to 3 heteoatoms selected from N, NR9, O or S, or 5 to 9
membered heterocycloalkyl having 1 or 2 heteroatoms selected from
N, NR9, O or S;
or a pharmaceutically acceptable salt thereof.
Preferred compounds of this invention include compounds of
structure B wherein both of the carbons of A adjacent to the
--NR.sup.5 -- group has a substituent other than hydrogen.
More preferred compounds of this invention include compounds of
structure B in which A is a phenyl wherein both of the carbons of A
adjacent to the --NR.sup.5 -- group has a substituent other than
hydrogen, and the carbon of group A para to the --NR.sup.5 -- group
has a substituent other than hydrogen.
More preferred compounds of this invention include compounds of
structure B in which A is a phenyl wherein:
both of the carbons of A adjacent to the --NR.sup.5 -- group has a
substituent other than hydrogen;
the carbon of group A para to the --NR.sup.5 -- group has a
substituent other than hydrogen; and
Y is a phenyl ring substituted at the 1- and 4-positions by X and
Z, respectively.
More preferred compounds of this invention include compounds of
structure B in which A is a phenyl wherein:
both of the carbons of A adjacent to the --NR.sup.5 -- group has a
substituent other than hydrogen;
the carbon of group A para to the --NR.sup.5 -- group has a
substituent other than hydrogen; and
Y is a phenyl ring substituted at the 1- and 4-positions by X and
Z, respectively;
and X is SO.sub.2.
More preferred compounds of this invention include compounds of
structure B in which A is a phenyl wherein:
both of the carbons of A adjacent to the --NR.sup.5 -- group has a
substituent other than hydrogen; and
the carbon of group A para to the --NR.sup.5 -- group has a
substituent other than hydrogen;
Y is a phenyl ring substituted at the 1- and 4-positions by X and
Z, respectively;
X is SO.sub.2 ;
and Z is oxygen.
More preferred compounds of this invention include compounds of
structure B in which A is a phenyl wherein:
both of the carbons of A adjacent to the --NR.sup.5 -- group has a
substituent other than hydrogen; and
the carbon of group A para to the --NR.sup.5 -- group has a
substituent other than hydrogen;
Y is a phenyl ring substituted at the 1- and 4-positions by X and
Z, respectively;
X is SO.sub.2 ;
Z is oxygen;
and R.sub.6 and R.sub.7 are hydrogen.
More preferred compounds of this invention include compounds of
structure B in which A is a phenyl wherein:
both of the carbons of A adjacent to the --NR.sup.5 -- group has a
substituent other than hydrogen;
the carbon of group A para to the --NR.sup.5 -- group has a
substituent other than hydrogen;
Y is a phenyl ring substituted at the 1- and 4-positions by X and
Z, respectively;
X is SO.sub.2 ;
Z is oxygen;
R.sub.6 and R.sub.7 are hydrogen;
and R.sub.8 is --CH.sub.2 OH or methyl.
Most preferred compounds of the present invention include
5-Bromo-2-{[4-(4-cyclobutylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-ami
no}-N-hydroxy-3-methyl-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(4-methylamino-but-2-ynyloxy)-benze
nesulfonyl]-amino}-benzamide;
5-Bromo-2-({4-[4-(3-dimethylamino-propylamino)-but-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-N-hydroxy-3-methyl-benzamide;
5-Bromo-2-({4-[4-(2-dimethylamino-ethylamino)-but-2-ynyloxy]-benzenesulfony
l}-methyl-amino)-N-hydroxy-3-methyl-benzamide;
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-methyl-bipheny-3-carbo
xylic acid hydroxyamide;
5-Bromo-N-hydroxy-3-methyl-2-[methyl-(4-prop-2-ynyloxy-benzenesulfonyl)-ami
no]-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-[methyl-(4-pent-2-ynyloxy-benzenesulfonyl)-ami
no]-benzamide;
5-Bromo-2-[(4-hept-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-met
hyl-benzamide;
5-Bromo-2-[(4-hex-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-meth
yl-benzamide;
5-Bromo-N-hydroxy-2-{[4-(4-methoxy-but-2-ynyloxy)-benzenesulfonyl]-methyl-a
mino}-3-methyl-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(3-phenyl-prop-2-ynyloxy)-benzenesu
lfonyl]-amino}-benzamide;
5-Bromo-N-hydroxy-2-({4-[3-(3-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide;
5-Bromo-N-hydroxy-2-({4-[3-(2-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide;
5-Bromo-N-hydroxy-2-({4-[3-(4-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide;
2-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-5-iodo-3-methy
l-benzamide;
2-[Benzyl-(4-but-2-ynyloxy-benzenesulfonyl)-amino]-N-hydroxy-3,5-dimethyl-b
enzamide;
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(4-pyrrolidin-1-yl-but-2-ynyloxy)-b
enzenesulfonyl]-amino}-benzamide;
5-Bromo-2-{[4-(4-diethylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino}
-N-hydroxy-3-methyl-benzamide;
5-Bromo-2-[(4-but-2-ynyloxy-benzenesulfonyl)-(4-methyl-piperazin-1-ylmethyl
)-amino]-N-hydroxy-3-methyl-benzamide;
5-Bromo-N-hydroxy-3-methyl-2-(methyl-{4-[4-(tetrahydro-pyran-2-yloxy)-but-2
-ynyloxy]-benzenesulfonyl}-amino)-benzamide;
5-Bromo-N-hydroxy-2-{[4-(4-hydroxy-but-2-ynyloxy)-benzenesulfonyl]-methyl-a
mino}-3-methyl-benzamide;
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-(4-methyl-piperazin-1-
ylmethyl)-biphenyl-3-carboxylic acid hydroxyamide dihydrochloride
salt; and
pharmaceutical salts thereof.
Heteroaryl, as used herein is a 5-10 membered mono- or bicyclic
aromatic ring having from 1-3 heteroatoms selected from N, NR9, S
and O. Heteroaryl is preferably ##STR9##
wherein K is NR9, O or S and R9 is hydrogen, phenyl, naphthyl,
alkyl of 1-6 carbon atoms, or cycloalkyl of 3-6 carbon atoms.
Preferred heteroaryl rings include pyrrole, furan, thiophene,
pyridine, pyrimidine, pyridazine, pyrazine, triazole, pyrazole,
imidazole, isothiazole, thiazole, isoxazole, oxazole, indole,
isoindole, benzofuran, benzothiophene, quinoline, isoquinoline,
quinoxaline, quinazoline, benzotriazole, indazole, benzimidazole,
benzothiazole, benzisoxazole, and benzoxazole. Heteroaryl groups of
the present invention may optionally be mono- or
di-substituted.
Heterocycloalkyl as used herein refers to a 5 to 10 membered
saturated or unsaturated mono or bi-cyclic ring having 1 or 2
heteroatoms selected from N, NR9, S or O. Heterocycloalkyl rings of
the present invention are preferably selected from ##STR10##
wherein K is NR9, O or S and R9 is hydrogen, phenyl, naphthyl,
alkyl of 1-6 carbon atoms, or cycloalkyl of 3-6 carbon atoms.
Preferred heterocycloalkyl rings include piperidine, piperazine,
morpholine, tetrahydropyran, tetrahydrofuran or pyrrolidine.
Heterocycloalkyl groups of the present invention may optionally be
mono- or di-substituted.
Aryl, as used herein refers to phenyl or naphthyl which may,
optionally be mono-, di- or tri-substituted.
Alkyl, alkenyl, alkynyl, and perfluoroalkyl include both straight
chain as well as branched moieties. Alkyl, alkenyl, alkynyl, and
cycloalkyl groups may be unsubstituted unsubstituted (carbons
bonded to hydrogen, or other carbons in the chain or ring) or may
be mono- or poly-substituted.
Halogen means bromine, chlorine, fluorine, and iodine.
Suitable substituents of aryl, heteroaryl, alkyl, alkenyl, alkynyl,
cycloalkyl and include, but are not limited to halogen, alkyl of
1-6 carbon atoms, alkenyl of 2-6 carbon atoms, alkynyl of 2-6
carbon atoms, cyclocalkyl of 3-6 carbon atoms, --OR.sub.2, --CN,
--COR.sub.2, perfluoroalkyl of 1-4 carbon atoms,
--O--perfluoroalkyl of 1-4 carbon atoms, --CONR.sub.2 R.sub.3,
--S(O).sub.n R.sub.2 --OPO(OR.sub.2)OR.sub.3,
--PO(OR.sub.2)R.sub.3, --OC(O)NR.sub.2 R.sub.3, --C(O)NR.sub.2
OR.sub.3, --COOR.sub.2, --SO.sub.3 H, --NR.sub.2 R.sub.3,
--N[(CH.sub.2).sub.2 ].sub.2 NR.sub.2, --NR.sub.2 COR.sub.3,
--NR.sub.2 COOR.sub.3, --SO.sub.2 NR.sub.2 R.sub.3, --NO.sub.2,
--N(R.sub.2)SO.sub.2 R.sub.3, --NR.sub.2 CONR.sub.2 R.sub.3,
--NR.sub.2 C(.dbd.NR.sub.3)NR.sub.2 R.sub.3, --NR.sub.2
C(.dbd.NR.sub.3)N(SO.sub.2)R.sub.2 R.sub.3, NR.sub.2
C(.dbd.NR.sub.3)N(C.dbd.O)R.sub.2 R.sub.3, NR.sub.2
C(.dbd.NR.sub.3)N(SO.sub.2 R.sub.2)R.sub.3, NR.sub.2
C(.dbd.NR.sub.3)N(COR.sub.2)R.sub.3, --SO.sub.2 NHCOR.sub.4,
--CONHSO.sub.2 R.sub.4, -tetrazol-5-yl, --SO.sub.2 NHCN, --SO.sub.2
NHCONR.sub.2 R.sub.3, phenyl, naphthyl, heteroaryl or
heterocycloalkyl;
wherein --NR.sub.2 R.sub.3 may form a pyrrolidine, piperidine,
morpholine, thiomorpholine, oxazolidine, thiazolidine,
pyrazolidine, piperazine, or azetidine ring;
R.sub.2 and R.sub.3 are each, independently, hydrogen, alkyl of 1-6
carbon atoms, cycloalkyl of 3-6 carbon atoms, phenyl, naphthyl,
heteroaryl or heterocycloalkyl;
R.sub.4 is alkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms,
alkynyl of 2-6 carbon atoms, cycloalkyl of 3-6 carbon atoms;
perfluoroalkyl of 1-4 carbon atoms, phenyl, naphthyl, heteroaryl or
heterocycloalkyl; and n is 0 to 2.
Suitable substituents of heterocycloalkyl groups of the present
invention include, but are not limited to alkyl of 1-6 carbon
atoms, cycloalkyl of 3-6 carbon atoms, phenyl, naphthyl, heteroaryl
and heterocycloalkyl.
When a moiety contains more than substituent with the same
designation each of those substituents may be the same or
different.
Pharmaceutically acceptable salts can be formed from organic and
inorganic acids, for example, acetic, propionic, lactic, citric,
tartaric, succinic, fumaric, maleic, malonic, mandelic, malic,
phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,
methanesulfonic, naphthalenesulfonic, benzenesulfonic,
toluenesulfonic, camphorsulfonic, and similarly known acceptable
acids when a compound of this invention contains a basic moiety.
Salts may also be formed from organic and inorganic bases,
preferably alkali metal salts, for example, sodium, lithium, or
potassium, when a compound of this invention contains an acidic
moiety.
The compounds of this invention may contain an asymmetric carbon
atom and some of the compounds of this invention may contain one or
more asymmetric centers and may thus give rise to optical isomers
and diastereomers. While shown without respect to stereochemistry,
the present invention includes such optical isomers and
diastereomers; as well as the racemic and resolved,
enantiomerically pure R and S stereoisomers; as well as other
mixtures of the R and S stereoisomers and pharmaceutically
acceptable salts thereof. It is recognized that one optical isomer,
including diastereomer and enantiomer, or stereoisomer may have
favorable properties over the other. Thus when disclosing and
claiming the invention, when one racemic mixture is disclosed, it
is clearly contemplated that both optical isomers, including
diastereomers and enantiomers, or stereoisomers substantially free
of the other are disclosed and claimed as well.
The compounds of this invention are shown to inhibit the enzymes
MMP-1, MMP-9, MMP-13 and TNF-.alpha. converting enzyme (TACE) and
are therefore useful in the treatment of arthritis, tumor
metastasis, tissue ulceration, abnormal wound healing, periodontal
disease, graft rejection, insulin resistance, bone disease and HIV
infection. In particular, the compounds of the invention provide
enhanced levels of inhibition of the activity of TACE in vitro and
in cellular assay and/or enhanced selectivity over MMP-1 and are
thus particularly useful in the treatment of diseases mediated by
TNF.
The invention is further directed to a process for making compounds
of structure B involving one or more reactions as follows:
1) alkylating a compound of formula I, or a salt or solvate
thereof, ##STR11##
into a compound of formula II ##STR12##
2) reacting a compound of formula II above, or a salt or solvate
thereof, with a chlorinating agent such as thionyl chloride,
chlorosulfonic acid, oxalyl chloride, phosphorus pentachloride, or
other halogenating agents such as fluorosulfonic acid or thionyl
bromide to a compound of formula III: ##STR13##
wherein J is fluorine, bromine, chlorine.
The resultant sulfonyl chloride, fluoride or bromide, may be
further converted into triazolide, imidazolide or benzothiazolide
derivatives, where J is 1,2,4-triazolyl, benzotriazolyl or
imidazol-yl, by reacting the compound with 1,2,4-triazole,
imidazole or benzotriazole, respectively. R.sub.6, R.sub.7 and
R.sub.8 are as defined above.
The invention is still further directed to a process for making
compounds of structure B involving one or more reactions as
follows:
1) alkylating phenol, or a salt or solvate thereof, into a compound
of formula IV: ##STR14##
2) reacting a compound of formula IV above, or a salt or solvate
thereof with chlorosulfonic acid to prepare a compound of formula
II above.
Particularly preferred intermediates are compounds of formulae II
and III, with the proviso that R6 is not hydrogen.
The invention compounds are prepared using conventional techniques
known to those skilled in the art of organic synthesis. The
starting materials used in preparing the compounds of the invention
are known, made by known methods or are commercially available.
Some of the starting materials and intermediates, and methods for
making said starting materials and intermediates are disclosed in
U.S. Pat. No. 5,776,961.
Those skilled in the art will recognize that certain reactions are
best carried out when other potentially reactive functionality on
the molecule is masked or protected, thus avoiding undesirable side
reactions and/or increasing the yield of the reaction. To this end,
those skilled in the art may use protecting groups. Examples of
these protecting group moieties may be found in T. W. Greene, P. G.
M. Wuts "Protective Groups in Organic Synthesis", 2.sup.nd Edition,
1991, Wiley & Sons, New York. Reactive side chain
functionalities on amino acid starting materials are preferably
protected. The need and choice of protecting groups for a
particular reaction is known to those skilled in the art and
depends on the nature of the functional group to be protected
(hydroxy, amino, carboxy, etc.), the structure and stability of the
molecule of which the substituent is part and the reaction
conditions.
When preparing or elaborating compounds of the invention containing
aryl, heteroaryl or heterocyclic rings, those skilled in the art
recognize that substituents on that ring may be prepared before,
after or concomitant with construction of the ring. For clarity,
substituents on such rings have been omitted from the schemes
herein below.
Those skilled in the art will recognize that the nature and order
of the synthetic steps presented may be varied for the purpose of
optimizing the formation of the compounds of the invention.
The hydroxamic acid compounds of the invention, 1, are prepared
according to Scheme 1 by converting a carboxylic acid, 2, into the
corresponding acid chloride or anhydride, or by reacting it with a
suitable peptide coupling reagent, followed by reaction with
hydroxylamine to give 1, or with a protected hydroxylamine
derivative to give 3. Compounds 3, wherein R.sub.30 is a t-butyl,
benzyl, trialkylsilyl or other suitable masking group may then be
deprotected by known methods to provide the hydroxamic acid 1.
##STR15##
Carboxylic acids 2 may be prepared as shown in Scheme 2. Amino acid
derivative 4, in which R.sub.40 is hydrogen or a suitable
carboxylic acid protecting group, may be sulfonylated or
phosphorylated by reacting with compounds 5, in which J is a
suitable leaving group including, but not limited to chlorine. The
N--H compound 6 may then be alkylated with R.sub.3 J and a base
such as potassium carbonate or sodium hydride in a polar aprotic
solvent such as acetone, N,N-dimethylformamide (DMF), or
tetrahydrofuran (THF) to provide sulfonamide 7. Compound 7 is also
available through direct reaction of 5 with an N-substituted amino
acid derivative, 8. Conversion of 7 into the carboxylic acid is
performed by acid, base hydrolysis, or other method consistent with
the choice of protecting group R.sub.40 and the presence of a
carbon-carbon triple bond. ##STR16##
Methods of preparation of sulfonylating agents 5 are shown in
Scheme 3. Thus, sulfonic acid salts 9, where ZR.sub.50 is a
hydroxy, thiol or substituted amino moiety may be alkylated with
acetylenes 10, where J is a suitable leaving group such as halogen
mesylate, tosylate, or triflate to give 11. Acetylenes 10 are
commercially available or known compounds, or they may be
synthesized by known methods by those skilled in the art. The
sulfonic acid salts 11 may be converted into the corresponding
sulfonyl chloride or other sulfonylating agent 5 by known methods,
such as reaction with oxalyl chloride or other reagent compatible
with substituents R.sub.6, R.sub.7 and R.sub.8 and the acetylene.
Alternatively, the disulfide 12 may be converted into di-acetylene
13 by reaction with compounds 10, followed by reduction of the
disulfide bond to provide the analogous thiols which may be
converted into 5 by known methods. Alkylation of the phenol,
thiophenol, aniline or protected aniline 14 with 10 to give 15,
followed by reaction with chlorosulfonic acid provide sulfonic
acids 16 which are readily converted into 5 with oxalyl chloride or
similar reagents. Thiophenols 17 are also precursors to S via
protection of the thiol, alkylation of ZH, where Z is O, N or S,
and deprotection of the sulfur followed by oxidation to the
sulfonic acid 16. ##STR17##
The phosphorus containing analogs of 8 may be prepared using
similar methodology, as shown in Scheme 4. ##STR18##
The acetylenic side chain may also be appended after sulfonylation
or phosphorylation of the amino acid derivative, as shown in Scheme
5. Thus, the amino acid derivatives 4 and 8 can be sulfonylated or
phosphorylated with compounds 20, where ZR.sub.50 is hydroxy or
protected hydroxy, thiol or amine, and, if necessary, alkylated
with R.sub.7 J as in Scheme 2, to give 21. Removal of the R.sub.50
masking group to give 22 and subsequent alkylation of the resulting
phenol, thiol or amine with 10 provides 7. In the case where
ZR.sub.50 is equal to OH, no deprotection step is required to give
22. ##STR19##
The propargylic amine analogs of 7 can be synthesized as shown in
Scheme 6 starting from the amino acid derivatives 4 and/or 8.
Sulfonylation or phosphorylation with para-nitro aryl compound 23,
for example 4-nitrobenzenesulfonyl chloride, followed by alkylation
with R.sub.5 J (for 4) using a base such as potassium carbonate or
sodium hydride in DMF provides 24. Reduction of the nitro moiety
with hydrogen and palladium on carbon, tin chloride or other known
method to give aniline 25 and subsequent alkylation with 10 then
provides 7. Aniline 25 may be derivatized with a suitable nitrogen
protecting group, such as t-butoxycarbonyl, to give 26 prior to
alkylation with 10 subsequent deprotection after the alkylation
step. ##STR20##
Acetylenic derivatives 7 are also accessible via the fluoro
compounds 27, readily prepared from the amino acid derivatives 4
and/or 8 by reaction with fluoraryl 26, as shown in Scheme 7.
Displacement of the fluorine of 27 in the presence of a base such
as sodium hydride with a masked hydroxy, thiol, or amino group
(HZR.sub.70, where R.sub.70 is a suitable protecting group) in a
polar aprotic solvent such as DMF, followed by deprotection gives
28, which can then be alkylated with 10 to provide 7. Conversion of
27 to 28, where Z is sulfur, might also be accomplished with
Na.sub.2 S, K.sub.2 S, NaSH or KS(C.dbd.S)OEt. The fluorine of 27
can also be displaced in a polar aprotic solvent with the
propargylic derivative 29, where Z is O, S or NH, in the presence
of a base such as sodium hydride, to give 7 directly. ##STR21##
Compound 7, wherein Z is a methylene group, is available via 30, as
shown in Scheme 8. Benzylic bromination of 30 with
N-bromosuccinimide in a chlorinated hydrocarbon solvent provides
bromide 31. This is followed by displacement of the bromide with
the appropriate propynyl cuprate to provide sulfonamide 8.
##STR22##
Compounds of the invention can also be prepared by modifying
substituents on the acetylenic side chain at any stage after
sulfonylation or phosphorylation of the starting amino acid
derivatives 4 or 8. Functional groups such as halogen, hydroxy,
amino, aldehyde, ester, ketone, etc. may be manipulated by standard
methods to form the moieties defined by R.sub.1 -R.sub.8 of
compounds 1. It is recognized by those skilled in the art of
organic synthesis that the successful use of these methods is
dependent upon the compatibility of substituents on other parts of
the molecule. Protecting groups and/or changes in the order of
steps described herein may be required.
Some of the methods available for the derivatization of compounds
of structure 32 (equivalent to compound 7 wherein R.sub.12 is
hydrogen) are shown in Scheme 9. Metallation of the terminal
acetylene 32 followed by addition of an aldehyde or alkyl halide,
sulfonate or triflate provides derivatives 33 and 34. Reaction of
32 with formaldehyde and an amine provides the Mannich addition
product 35. Cyanogen bromide addition to 35 gives the propargylic
bromide 36 which may be displaced with a variety of nucleophiles to
give, for example, ethers, thioethers and amines 37. Palladium
catalyzed coupling reactions of 32 provide the aryl or heteroaryl
acetylenes 38. It is recognized by those skilled in the art of
organic synthesis that the successful use of these methods is
dependent upon the compatibility of substituents on other parts of
the molecule. Protecting groups and/or changes in the order of
steps described herein may be required. ##STR23##
The following specific examples illustrate the preparation of
representative compounds of this invention. The starting materials,
intermediates, and reagents are either commercially available or
can be readily prepared following standard literature procedures by
one skilled in the art of organic synthesis.
EXAMPLE 1
3-Methyl-5-bromo-2-(4-fluoro-benzenesulfonylamino)-benzoic acid
To a solution of 25.0 g (0.102 mol) of
2-amino-3-methyl-5-bromobenzoic acid methyl ester (U.S. Pat. No.
5,776,961) in 300 mL of pyridine was added 21.93 g (0.113 mol) of
4-fluorobenzenesulfonyl chloride. The reaction mixture was stirred
for 18 h at 80.degree., cooled to room temperature and poured into
water. The resulting mixture was extracted with ethyl acetate and
the combined organics were then washed with 5% HCl solution and
water. The organic layer was then dried over MgSO.sub.4, filtered
and concentrated in vacuo. The residue was chromatographed on
silica gel eluting with ethyl acetate/hexanes (1:3) to provide
14.53 g (39%) of the desired product as a tan solid. Electrospray
Mass Spec: 388.0 (M-H).sup.-
EXAMPLE 2
5-Bromo-2-[(4-fluoro-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid methyl ester
To a solution of 14.53 g (0.040 mol) of the product of Example 1 in
300 mL of DMF was added 66.1 g (0.479 mol) of potassium carbonate
followed by 9.94 mL (0.160 mol) of iodomethane. The resulting
mixture was stirred at room temperature for 48 h and then diluted
with water and ether. The organic layer was separated and washed
with water and then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was chromatographed on silica
gel eluting with ethyl acetate/hexanes (1:10) to provide 12.83 g
(82%) of the desired product as a pale yellow solid. Electrospray
Mass Spec: 415.8 (M+H).sup.+
EXAMPLE 3
5-Bromo-2-[(4-hydroxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid
To a solution of 17.0 mL (0.227 mol) of 2-butyn-1-ol in 375 mL of
DMF at room temperature was added 9.08 g (0.227 mol) of 60% sodium
hydride. The resulting mixture was stirred for 0.5 h and then a
solution of 17.8 g (0.045 mol) of the product of Example 2
dissolved in 50 mL of DMF was added to the reaction. The reaction
mixture was then heated to reflux for 24 h, cooled to room
temperature and then acidified to pH 2 with 10% HCl solution. After
stirring for 1 h the resulting mixture was diluted with water and
extracted with ethyl acetate. The combined organics were then dried
over MgSO.sub.4, filtered and concentrated in vacuo. The residue
was chromatographed on silica gel eluting with ethyl
acetate/hexanes (1:3) to provide 12.5 g (69%) of the desired
phenol-carboxylic acid product as a white solid. Electrospray Mass
Spec: 401.8 (M+H).sup.+
EXAMPLE 4
5-Bromo-2-[(4-hydroxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid methyl ester
To a solution of 15.2 g (0.038 mol) of the product of Example 3 in
125 mL of DMF was added 9.58 g (0.114 mol) of sodium bicarbonate
followed by 4.7 mL (0.076 mol) of iodomethane. The resulting
mixture was stirred at room temperature for 5 h and then diluted
with ether and water. The organics were separated and washed with
water, dried over MgSO.sub.4, filtered and concentrated in vacuo.
The residue was chromatographed on silica gel eluting with ethyl
acetate/hexanes (1:3) to provide 12.27 g (78%) of the desired
phenol-ester product as a pale yellow solid. Electrospray Mass
Spec: 413.7 (M+H).sup.+
EXAMPLE 5
5-Bromo-3-methyl-2-[methyl-(4-prop-2-ynyloxy-benzenesulfonyl)-amino]-benzoi
c acid methyl ester
To a solution of 0.317 g (1.208 mmol) of triphenylphosphine
dissolved in 5 mL of benzene and 2 mL of THF was added 0.070 mL
(1.208 mmol) of propargyl alcohol. After five minutes 0.500 g
(1.208 mmol) of the product of Example 4, dissolved in 2 mL of THF,
was added to the reaction followed by 0.190 mL (1.208 mmol) of
diethyl azodicarboxylate. The resulting reaction mixture was
stirred for 18 h at room temperature and then concentrated in
vacuo. The residue was chromatographed on silica gel eluting with
ethyl acetate/hexanes (1:10) to provide 0.389 g (71%) of the
desired propargylic ether as a white solid. Electrospray Mass Spec:
451.8 (M+H).sup.+
EXAMPLE 6
5-Bromo-2-[(4-but-2-ynyloxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid
According to the procedure of Example 5 0.150 g (0.363 mmol) of the
product of Example 4 was reacted with 0.027 mL (0.362 mmol) of
2-butyn-1-ol to give 0.106 g (63%) of the alkynyloxy ether.
The alkynyloxy ether was dissolved in 2.2 mL of THF/methanol (1:1)
and 1.1 mL of 1.0N sodium hydroxide solution was added. The
reaction was heated to reflux overnight, cooled to room temperature
and acidified with 10% HCl solution. The mixture was extracted with
ethyl acetate and the combined organics were dried over MgSO.sub.4,
filtered and concentrated in vacuo to provide 0.099 g (97%) of the
desired carboxylic acid product as a white solid. Electrospray Mass
Spec: 451.8 (M+H).sup.+
EXAMPLE 7
5-Bromo-3-methyl-2-[methyl-(4-pent-2-ynyloxy-benzenesulfonyl)-amino]-benzoi
c acid
According to the procedure of Example 6 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.056 mL (0.604 mmol) of
2-pentyn-1-ol to give 0.233 g of the ether-ester, followed by base
hydrolysis to give 0.22 g (97%) of the carboxylic acid as a white
solid. Electrospray Mass Spec: 465.9 (M+H).sup.+
EXAMPLE 8
5-Bromo-2-[(4-hept-2-ynyloxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoi
c acid
According to the procedure of Example 6 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.077 mL (0.604 mmol) of
2-heptyn-1-ol to give 0.246 g of the ether-ester, followed by base
hydrolysis to give 0.214 g (90%) of the carboxylic acid as a white
solid. Electrospray Mass Spec 494.0 (M+H).sup.+
EXAMPLE 9
5-Bromo-2-[(4-hex-2-ynyloxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid methyl ester
According to the procedure of Example 5 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.059 g (0.604 mmol) of
2-hexyn-1-ol to give 0.206 g (69%) of the alkynyloxy ether.
Electrospray Mass Spec 494.0 (M+H).sup.+
EXAMPLE 10
5-Bromo-2-[(4-hex-2-ynyloxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid
To a solution of 0.206 g (0.417 mmol) of the product of Example 9
dissolved in 4.0 mL of THF/methanol (1:1) and 0.5mL of 5.0N sodium
hydroxide solution was added. The reaction was stirred overnight at
room temperature and then acidified with 10% HCl solution. The
mixture was extracted with dichloromethane and the combined
organics were dried over MgSO.sub.4, filtered and concentrated in
vacuo to provide a white solid which was washed with ether/hexanes
(1:1) and dried in vacuo to give 0.163 g (82%) of the desired
carboxylic acid product as a white solid. Electrospray Mass Spec:
477.9 (M-H).sup.-
EXAMPLE 11
5-Bromo-3-methyl-2-{methyl-[4-(3-phenyl-prop-2-ynyloxy)-benzenesulfonyl]-am
ino}-benzoic acid methyl ester
According to the procedure of Example 5 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.080 g (0.604 mmol) of
3-phenyl propargyl alcohol to give 0.272 g (85%) of the alkynyloxy
ether as a brown oil. Electrospray Mass Spec 527.8 (M+H).sup.+
EXAMPLE 12
5-Bromo-2-({4-[3-(3-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid methyl ester
According to the procedure of Example 5 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.098 g (0.604 mmol) of
3-(3-methoxy)-phenyl propargyl alcohol to give 0.285 g (85%) of the
alkynyloxy ether as a pale yellow oil. Electrospray Mass Spec 557.8
(M+H).sup.+
EXAMPLE 13
5-Bromo-2-({4-[3-(2-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid methyl ester
According to the procedure of Example 5 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.098 g (0.604 mmol) of
3-(2-methoxy)-phenyl propargyl alcohol to give 0.296 g (88%) of the
alkynyloxy ether as a colorless oil. Electrospray Mass Spec 557.8
(M+H).sup.+
EXAMPLE 14
5-Bromo-2-({4-[3-(4-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid methyl ester
According to the procedure of Example 5 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.098 g (0.604 mmol) of
3-(4-methoxy)-phenyl propargyl alcohol to give 0.240 g (71%) of the
alkynyloxy ether as a brown oil. Electrospray Mass Spec 557.8
(M+H).sup.+
EXAMPLE 15
5-Bromo-3-methyl-2-(methyl-{4-[4-(tetrahydro-pyran-2-yloxy)-but-2-ynyloxy]-
benzenesulfonyl}-amino)-benzoic acid methyl ester
According to the procedure of Example 5 0.500 g (1.208 mmol) of the
product of Example 4 was reacted with 0.226 g (1.328 mmol) of
4-tetrahydropyran-2-butyn-1,4-diol to give 0.60 g (88%) of the
alkynyloxy ether as a colorless oil. Electrospray Mass Spec 567.9
(M+H).sup.+
EXAMPLE 16
5-Bromo-2-{[4-(tert-butyl-dimethyl-silanyloxy)-benzenesulfonyl]-methyl-amin
o}-3-methyl-benzoic acid methyl ester
To a solution of 1.25 g (3.019 mmol) of the product of Example 4 in
5.0 mL of DMF was added 0.514 g (7.548 mmol) of imidazole followed
by 0.546 g (3.623 mmol) of t-butyldimethylsilyl chloride. The
resulting mixture was stirred at room temperature for 15 h and then
diluted with ether and water. The organics were washed with water,
dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo.
The residue was chromatographed on silica gel eluting with ethyl
acetate/hexanes (1:10) to provide 1.34 g (84%) of the silyl ether
as a white solid. Electrospray Mass Spec 527.8 (M+H).sup.+
EXAMPLE 17
2-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-iodo-3-methyl-benzoic
acid methyl ester
To a solution of 0.340 g (0.644 mmol) of the product of Example 16
in 30 mL of dioxane was added 1.30 mL (2.576 mmol) of
bis(tributyltin) followed by 0.059 g (0.051 mmol) of
tetrakis(triphenylphosphine)palladium(0). The resulting mixture was
heated to reflux overnight and then concentrated in vacuo. The
residue was chromatographed on silica gel eluting with ethyl
acetate/hexanes (1:20) to provide 0.34 g (72%) of the desired aryl
stannane.
The stannane was then dissolved in 50 mL of chloroform and 10 mL of
a 0.1M solution of iodine in chloroform was added dropwise. After
15 minutes at room temperature the reaction was quenched with a
solution of 1.5 g of potassium fluoride in 30 mL of methanol
followed by 30 mL of 5% sodium bisulfite solution. The organic
layer was separated, and the aqueous layer was extracted with
dichloromethane. The combined organics were dried over Na.sub.2
SO.sub.4, filtered and concentrated in vacuo. The residue was
partitioned between hexanes and acetonitrile and the acetonitrile
layer was then concentrated in vacuo to give 0.265 g (100%) of the
crude aryl iodide.
The aryl iodide was dissolved in 5 mL of THF and 1.0 mL of a 1.0M
solution of tetrabutylammonium fluoride in THF was added. The
reaction was stirred for 1 h at room temperature and the acidified
with 5% HCl solution and extracted with dichloromethane. The
combined organics were dried over MgSO.sub.4, filtered and
concentrated in vacuo to provide 0.185 g (87%) of the phenol.
According to the procedure of Example 5 0.185 g (0.401 mmol) of the
phenol and 0.030 mL of 2-butyn-1-ol provided 0.143 g (69%) of the
desired ether as a colorless oil. Electrospray Mass Spec 513.8
(M+H).sup.+
EXAMPLE 18
2-[Benzyl-(4-hydroxy-benzenesulfonyl)-amino]-3,5-dimethyl-benzoic
acid
According to the procedure of Example 3 1.360 g (3.185 mmol) of
2-[benzyl-(4-fluoro-benzenesulfonyl)-amino]-3,5-dimethyl-benzoic
acid methyl ester (U.S. Pat. No. 5,776,961) gives 0.956 g (73%) of
the phenol-carboxylic acid as a white solid after trituration with
ether. Electrospray Mass Spec 411.9 (M+H).sup.+
EXAMPLE 19
2-[Benzyl-(4-hydroxy-benzenesulfonyl)-amino]-3,5-dimethyl-benzoic
acid methyl ester
To a solution of 0.820 g (1.995 mmol) of the product of Example 18
in 10 mL of DMF was added 0.826 g (5.985 mmol) of potassium
carbonate followed by 0.124 mL (1.995 mmol) of iodomethane. The
reaction was stirred at room temperature for 2 h and then diluted
with ether and water and acidified with 5% HCl solution. The
Aqueous layer was extracted with ethyl acetate and the combined
organics were dried over MgSO.sub.4, filtered and concentrated in
vacuo. The residue was chromatographed on silica gel eluting with
ethyl acetate/hexanes (1:3) to provide 0.090 g (87%) of the
phenol-ester. Electrospray Mass Spec 425.9 (M+H).sup.+
EXAMPLE 20
2-[Benzyl-(4-but-2-ynyloxy-benzenesulfonyl)-amino]-3,5-dimethyl-benzoic
acid methyl ester
According to the procedure of Example 5 0.250 g (0.588 mmol) of the
product of Example 19 was reacted with 0.44 mL (0.588 mmol) of
2-butyn-1 ol to give 0.203 g (72%) of the alkynyloxy ether as a
white solid. Electrospray Mass Spec 477.9 (M+H).sup.+
EXAMPLE 21
5-Bromo-2-[(4-hydroxy-benzenesulfonyl)-(4-methyl-piperazin-1-ylmethyl)-amin
o]-3-methyl-benzoic acid methyl ester
To a solution of 1.0 g (1.894 mmol) of the product of Example 16 in
60 mL of carbon tetrachloride was added 0.405 g of
N-bromosuccinimide. The resulting mixture was heated to reflux for
2 h while being irradiated by a sun lamp. The reaction was then
cooled top room temperature, washed with water, dried over Na.sub.2
SO.sub.4, filtered and concentrated in vacuo.
The residue was dissolved in 5.0 mL of DMF and 0.784 g (5.682 mmol)
of potassium carbonate was added followed by 0.21 mL (1.894 mmol)
of 1-methylpiperazine. The resulting mixture was stirred for 48 h
at room temperature and then diluted with water and ethyl acetate.
The organics were washed with water and then extracted with 10% HCl
solution. The acid layer was neutralized with 1.0N NaOH solution
and extracted with dichloromethane. The combined dichloromethane
extracts were dried over Na.sub.2 SO.sub.4, filtered and
concentrated in vacuo to provide 0.501 g (52%) of the
piperazine-phenol as a white solid. Electrospray Mass Spec 511.9
(M+H).sup.+
EXAMPLE 22
5-Bromo-2-
[(4-but-2-ynyloxy-benzenesulfonyl)-methyl-amino]-3-(4-methyl-piperazin-1-y
lmethyl)-benzoic acid methyl ester
To a solution of 0.963 g (3.672 mmol) of triphenylphosphine in 5.0
mL of THF was added 0.275 mL (3.672 mmol) of 2-butyn-1-ol followed
by 0.376 g (0.734 mmol) of the product of Example 21 dissolved in 2
mL of dichloromethane. After 5 minutes at room temperature 0.578 mL
(3.672 mmol) of diethyl azodicarboxylate was added dropwise and the
resulting reaction mixture was stirred overnight at room
temperature and then concentrated in vacuo. The residue was diluted
with ethyl acetate and the organics were extracted with 10% HCl
solution. The combined acid extracts were basified with 1.0N NaOH
solution and then extracted with dichloromethane. The combined
dichloromethane extracts were dried over Na.sub.2 SO.sub.4,
filtered and concentrated in vacuo. The residue was chromatographed
on silica gel eluting with dichloromethane/methanol/triethylamine
(100:2:0.5) to provide 0.149 g (36%) of the butynyl ether as a pale
yellow oil. Electrospray Mass Spec 563.9 (M+H).sup.+
EXAMPLE 23
5-Bromo-3-methyl-2-{methyl-[4-(4-pyrrolidin-1-yl-but-2-ynyloxy)-benzenesulf
onyl]-aniino}-benzoic acid methyl ester
To a solution of 0.300 g (0.664 mmol) of the product of Example 5
in 2.0 mL of dioxane was added 0.054 g (1.659 mmol) of
paraformaldehyde, 0.111 mL (1.327 mmol) of pyrrolidine, 0.245 mL of
acetic acid and 2.5 mg of cuprous chloride. The resulting mixture
was stirred at room temperature for 15 minutes and then heated to
reflux for 2 h. After cooling to room temperature the reaction
mixture was extracted with 10% HCl solution and the aqueous
extracts were then basified with 1.0N NaOH solution and extracted
with ether. The combined ether extracts were dried over Na.sub.2
SO.sub.4, filtered and concentrated in vacuo to provide 0.216 g
(61%) of the pyrrolidine-alkyne as a brown oil. Electrospray Mass
Spec 520.9 (M+H).sup.+
EXAMPLE 24
5-Bromo-2-{[4-(4-diethylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino}
-3-methyl-benzoic acid methyl ester
According to the procedure of Example 23 1.00 g (2.212 mmol) of the
product of Example 5 provides 0.749 g (63%) of the
diethylamino-alkyne as a yellow oil after chromatography on silica
gel eluting with ethyl acetate. Electrospray Mass Spec 536.9
(M+H).sup.+
EXAMPLE 25
5-Bromo-2-{[4-(4-bromo-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino}-3-meth
yl-benzoic acid methyl ester
To a 0.degree. solution of 0.707 g (1.317 mmol) of the product of
Example 24 dissolved in 10 mL of ether was added 0.53 mL of a 3.0M
solution of cyanogen bromide in dichloromethane. The reaction was
warmed to room temperature and stirred overnight. The reaction
mixture was then filtered and the filtrate was diluted with ether,
washed with 5% HCl solution and brine. The organics were dried over
Na.sub.2 SO.sub.4, filtered and concentrated in vacuo to provide
0.628 g (88%) of the propargylic bromide as a light brown oil.
EXAMPLE 26
5-Bromo-2-{[4-(4-methoxy-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino}-3-me
thy-l-benzoic acid
To a solution of 0.117 g (0.215 mmol) of the product of Example 25
in 1.0 mL of THF and 3.0 mL of methanol was added 0.5 mL of a 5.0N
solution of sodium hydroxide followed by 3.6 mg of
tetrabutylammonium hydrogen sulfate. The resulting reaction mixture
was stirred at room temperature for 15 h and then acidified with
10% HCl solution and extracted with dichloromethane. The combined
organics were dried over MgSO.sub.4, filtered and concentrated in
vacuo to provide 0.083 g (81%) of the bis-propargylic ether
carboxylic acid as a white solid. Electrospray Mass Spec 481.8
(M+H).sup.+
EXAMPLE 27
5-Bromo-2-{[4-(4-cyclobutylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-ami
no}-3-methyl-benzoic acid methyl ester
To a solution of 0.300 g (0.550 mmol) of the product of Example 25
in 3.0 mL of THF was added 0.103 mL (1.211 mmol) of cyclobutylamine
and the reaction was stirred for 15 h at room temperature. The
resulting mixture was diluted with ether and the organics were
washed with water and brine, dried over Na.sub.2 SO.sub.4, filtered
and concentrated in vacuo. The residue was chromatographed on
silica gel eluting with a gradient of ethyl acetate hexanes (1:1)
to chloroform/methanol (9:1) to provide 0.199 g (69%) of the
propargylic cyclobutylamine as a colorless oil. Electrospray Mass
Spec 535.0 (M+H).sup.+
EXAMPLE 28
(4-{4-[(4-Bromo-2-hydroxycarbamoyl-6-methyl-phenyl)-methyl-sulfamoyl]-pheno
xy}-but-2-ynyl)-cyclobutyl-carbamic acid tert-butyl ester
To a solution of 0.167 g (0.312 mmol) of the product of Example 27
in 2.0 mL of DMF was added 0.075 g (0.343 mmol) of di-t-butyl
dicarbonate and 7.6 mg of 4-dimethylaminopyridine. The reaction was
stirred for 15 h at room temperature and then diluted with ether
and washed with water, 5% HCl solution and brine. The organics were
then dried over Na.sub.2 SO.sub.4, filtered and concentrated in
vacuo to provide 0.173 g (87%) of the carbamate.
To a solution of 0.173 g (0.272 mmol) of the t-butyl carbamate
dissolved in 6.0 mL of THF/methanol (1:1) was added 1.4 mL of a
1.0N NaOH solution and the reaction mixture was then heated to
reflux for 15 h. After cooling to room temperature the reaction
mixture was acidified with 5% HCl solution and extracted with
dichloromethane. The combined organics were dried over MgSO.sub.4,
filtered and concentrated in vacuo. The residue was chromatographed
on silica gel eluting with ethyl acetate/hexanes (1:1) to provide
0.149 g (88%) of the carboxylic acid as a white solid.
To a 0.degree. solution of 0.040 mL (0.792 mmol) of a 2.0M solution
of oxalyl chloride in dichloromethane, dissolved in 3.4 mL of
dichloromethane, was added 0.061 mL (0.792 mmol) of DMF and the
mixture was stirred at 0.degree. for 15 minutes. A solution of
0.164 g of the carboxylic acid, dissolved in 1 mL of DMF, was then
added and the reaction was warmed to room temperature. The
resulting mixture is stirred for 1 h at room temperature and then
poured into a 0.degree. mixture of 0.8 mL of water, 4.0 mL of THF
and 0.25 mL of a 50% aqueous solution of hydroxylamine. The
reaction is allowed to warm to room temperature overnight and the
organics are then concentrated in vacuo. The residue is diluted
with ethyl acetate, washed with water and saturated sodium
bicarbonate, dried over Na.sub.2 SO.sub.4, filtered and
concentrated in vacuo to provide 0.153 g (91%) of the
carbamate-hydroxamic acid as a white foam. Electrospray Mass Spec
637.9 (M+H).sup.+
EXAMPLE 29
5-Bromo-3-methyl-2-{methyl-[4-(4-methylamino-but-2-ynyloxy)-benzenesulfonyl
]-amino}-benzoic acid methyl ester
According to the procedure of Example 27 0.325 g (0.596 mmol) of
the product of Example 25 reacted with methylamine to provide 0.132
g (45%) of the propargylic methylamine as a brown oil. Electrospray
Mass Spec 495.0 (M+H).sup.+
EXAMPLE 30
(4-{4-[(4-Bromo-2-hydroxycarbamoyl-6-methyl-phenyl)-methyl-sulfamoyl]-pheno
xy}-but-2-ynyl)-methyl-carbamic acid tert-butyl ester
According to the procedure of Example 28 0.110 g (0.222 mmol) of
the product of Example 29 was converted into 0.090 g of the
carbamate-hydroxamic acid. Electrospray Mass Spec 598.0
(M+H).sup.+
EXAMPLE 31
5-Bromo-2-{[4-(4-cyclobutylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-ami
no}-N-hydroxy-3-methyl-benzamide
To a solution of 0.100 g (0.157 mmol) of the product of Example 28
in 2.0 mL of dichloromethane was added 1.0 mL of trifluoroacetic
acid. The resulting solution was stirred at room temperature for 1
h and then concentrated in vacuo. The residue was diluted with
dichloromethane and washed with saturated sodium bicarbonate
solution, dried over Na.sub.2 SO.sub.4, filtered and concentrated
in vacuo to provide 0.083 g (99%) of the amino-hydroxamic acid.
To a solution of 0.078 g (0.146 mmol) of the amino-hydroxamic acid
dissolved in 2.0 mL of dichloromethane was added 0.29 mL (0.29
mmol) of a 1.0M solution of HCl in ether. The resulting mixture was
stirred for 1 h at room temperature and then diluted with ether.
The precipitate was filtered, washed with ether and dried in vacuo
to provide 0.068 g (82%) of the hydrochloride salt of the
amino-hydroxamic acid as a tan solid. Electrospray Mass Spec 535.8
(M+H).sup.+
EXAMPLE 32
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(4-methylamino-but-2-ynyloxy)-benze
nesulfonyl]-amino}-benzamide
According to the procedure of Example 31 0.066 g (0.111 mmol) of
the product of Example 30 provides 0.042 g of the desired
hydrochloride salt of the amino-hydroxamic acid. Electrospray Mass
Spec 495.8 (M+H).sup.+
EXAMPLE 33
5-Bromo-2-({4-[4-(3-dimethylamino-propylamino)-but-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-N-hydroxy-3-methyl-benzamide
According to the procedure of Example 27 0.300 g (0.550 mmol) of
the product of Example 25 reacted with 0.362 mL (2.75 mmol) of
dimethylamino propylamine to provide 0.076 g (24%) of the
propargylic diamine-ester.
According to the procedure of Example 28 0.135 g (0.239 mmol) of
the diamine-ester was converted to the t-butyl carbamate, followed
by hydrolysis of the ester and conversion of the carboxylic acid
into 0.059 g of the carbamate-hydroxamic acid.
According to the procedure of Example 31 0.058 g of the
carbamatehydroxamic acid was converted into 0.037 g of the desired
bis-hydrochloride salt of the diamino-hydroxamic acid, obtained as
a brown solid. Electrospray Mass Spec 569.0 (M+H).sup.+
EXAMPLE 34
5-Bromo-2-({4-[4-(2-dimethylamino-ethylamino)-but-2-ynyloxy]-benzenesulfony
l}-methyl-amino)-N-hydroxy-3-methyl-benzamide
According to the procedure of Example 27 0.300 g (0.550 mmol) of
the product of Example 25 reacted with 0.302 mL (2.75 mmol) of
N,N-dimethylethylenediamine to provide 0.114 g (38%) of the
propargylic diamine-ester.
According to the procedure of Example 28 0.192 g (0.348 mmol) of
the diamine-ester was converted to the t-butyl carbamate, followed
by hydrolysis of the ester and conversion of the carboxylic acid
into 0.073 g of the carbamate-hydroxamic acid.
According to the procedure of Example 31 0.057 g of the
carbamatehydroxamic acid was converted into 0.040 g of the desired
bis-hydrochloride salt of the diamino-hydroxamic acid, obtained as
a brown solid. Electrospray Mass Spec 553.0 (M+H).sup.+
EXAMPLE 35
5-Bromo-2-[(4-methoxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid methyl ester
To a solution of 5.00 g (0.012 mol) of
5-bromo-2-(4-methoxy-benzenesulfonylamino)-3-methyl-benzoic acid
methyl ester (U.S. Pat. No. 5,776,961) in 40 mL of DMF was added
0.604 g (0.015 mol) of 60% sodium hydride. The resulting mixture
was stirred at room temperature foe 0.5 h and then 1.2 ml (0.018
mol) of iodomethane was added. The reaction was then stirred for 15
h and then diluted with ether. The organics were washed with water,
dried over MgSO.sub.4, filtered and concentrated in vacuo. The
residue was triturated with ether and the resulting white solid was
collected by filtration and dried in vacuo to provide 4.45 g (86%)
of the N-methyl sulfonamide. Electrospray Mass Spec 429.8
(M+H).sup.+
EXAMPLE 36
5-Bromo-N-hydroxy-2-[(4-methoxy-benzenesulfonyl)-methyl-amino]-3-methyl-ben
zamide
To a solution of 0.200 g (0.467 mmol) of the product of Example 35
in 6.0 mL of THF/methanol (1:1) was added 2.3mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was triturated with
ether/hexanes (1:1) and the solid was collected and dried in vacuo
to provide 0.136 g (70%) of the desired carboxylic acid product as
a white solid.
To a 0.degree. solution of 0.036 mL (0.710 mmol) of a 2.0M solution
of oxalyl chloride in dichloromethane, diluted with 3.1 mL of
dichloromethane, is added 0.055 mL (0.710 mmol) of DMF and the
reaction is stirred for 15 minutes at 0.degree.. A solution of
0.098 g (0.237 mmol) of the carboxylic acid, dissolved in 1 mL of
DMF, was added to the reaction and the resulting mixture is stirred
for 1 h at room temperature and then poured into a 0.degree.
mixture of 0.7 mL of water, 3.6 mL of THF and 0.23 mL of a 50%
aqueous solution of hydroxylamine. The reaction is allowed to warm
to room temperature overnight and the organics are then
concentrated in vacuo. The residue is diluted with ethyl acetate,
washed with 5% HCl solution, water and saturated sodium
bicarbonate, dried over over Na.sub.2 SO.sub.4, filtered and
concentrated in vacuo to provide 0.081 g (79%) of the hydroxamic
acid as a white foam. Electrospray Mass Spec 428.8 (M+H).sup.+
EXAMPLE 37
5-Bromo-2-[(4-butoxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid methyl ester
According to the procedure of Example 5 0.250 g (0.604 mmol) of the
product of Example 4 was reacted with 0.055 mL (0.604 mmol) of
n-butanol to give 0.241 g (85%) of the n-butyl ether. Electrospray
Mass Spec 469.8 (M+H).sup.+
EXAMPLE 38
5-Bromo-2-[(4-butoxy-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic
acid
To a solution of 0.204 g (0.434 mmol) of the product of Example 37
in 6.0 mL of THF/methanol (1:1) was added 2.2mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was triturated with
ether/hexanes (1:1) and the solid was collected and dried in vacuo
to provide 0.206 g (104%) of the desired carboxylic acid product as
a white solid. Electrospray Mass Spec: 455.8 (M+H).sup.+
EXAMPLE 39
5-Bromo-2-[(4-butoxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-methyl-benz
amide
To a 0.degree. solution of 0.54 mL (1.072 mmol) of a 2.0M solution
of oxalyl chloride in dichloromethane, diluted with 4.7 mL of
dichloromethane, is added 0.083 mL (1.072 mmol) of DMF and the
reaction is stirred for 15 minutes at 0.degree. C. A solution of
0.163 g (0.357 mmol) of the carboxylic acid product of Example 38,
dissolved in 1 mL of DMF, was added to the reaction and the
resulting mixture is stirred for 1 h at room temperature and then
poured into a 0.degree. mixture of 1.0 mL of water, 5.4 mL of THF
and 0.34 mL of a 50% aqueous solution of hydroxylamine. The
reaction is allowed to warm to room temperature overnight and the
organics are then concentrated in vacuo. The residue is diluted
with ethyl acetate, washed with 5% HCl solution, water and
saturated sodium bicarbonate, dried over Na.sub.2 SO.sub.4,
filtered and concentrated in vacuo to provide 0.157 g (93%) of the
hydroxamic acid as a white foam. Electrospray Mass Spec 506.8
(M+H).sup.+
EXAMPLE 40
4-Amino-5-methyl-biphenyl-3-carboxylic acid methyl ester
To a solution of 5.0 g (0.021 mol) of
2-amino-5-bromo-3-methyl-benzoic acid methyl ester and 2.8 g (0.023
mol) of phenyl boronic acid in 150 mL of dimethoxyethane was added
20.5 mL of a 2.0M aqueous solution of Na.sub.2 CO.sub.3 and 1.18 g
(1.02 mmol) of Pd(PPh.sub.3).sub.4. The reaction mixture was
evacuated and filled with N.sub.2 three times and was then heated
to reflux overnight. The reaction was then cooled to room
temperature, and poured into ethyl acetate and water. The aqueous
layer was extracted three times with ethyl acetate. The combined
organics were washed with brine, dried over MgSO.sub.4 and
concentrated in vacuo. The residue was chromatographed on silica
gel using gradient elution (100% hexane to 4/1 hexane/ethyl
acetate) to provide 2.5 g (50%) of
4-amino-5-methyl-biphenyl-3-carboxylic acid methyl ester.
Electrospray Mass Spec: 241.8 (M+H).sup.+
EXAMPLE 41
5-Methyl-4-[4-(pyridin-4-yloxy)-benzenesulfonylamino]-biphenyl-3-carboxylic
acid methyl ester
To a solution of 1.43 g (5.93 mmol) of the product of Example 40 in
20 mL of pyridine was added 1.98 g (6.52 mmol)
4-(pyridin-4-yloxy)-benzenesulfonyl chloride and the reaction was
stirred overnight. Additional 4-(pyridin-4-yloxy)-benzenesulfonyl
chloride (0.5 g, 1.6 mmol) was added twice over the next two days.
The reaction was then diluted with water and extracted three times
with dichloromethane. The organics were combined, washed with
brine, dried over Na.sub.2 SO.sub.4, concentrated in vacuo and
chromatographed on silica gel using gradient elution (100% hexane
to 100% ethyl acetate) to provide
5-methyl-4-[4-(pyridin-4-yloxy)-benzenesulfonylamino]-biphenyl-3-carboxyli
c acid methyl ester 2.54 g (90%) as a white solid. Electrospray
Mass Spec: 475.2 (M+H).sup.+
EXAMPLE 42
4-[(4-Hydroxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-carboxylic
acid methyl ester
To a 0.degree. C. solution of 1.25 g (2.63 mmol) of the product of
Example 41 in 7 mL of DMF at was added 0.132 g (3.29 mmol) of 60%
sodium hydride. The reaction was held at 0.degree. C. for 15
minutes and then warmed to room temperature. Iodomethane (0.49 mL,
7.89 mmol) was added and the reaction was stirred overnight. The
reaction mixture was quenched by the addition of water and the
aqueous layer was extracted three times with dichloromethane. The
organics were combined, washed with water and brine, dried over
MgSO.sub.4, and concentrated in vacuo to provide 0.9 g (68%) of
5-methyl-4-[4-(1-methylpyridinium-4-oxy-benzenesulfonyl)-methyl-amino]-bip
henyl-3-carboxylic acid methyl ester iodide as a yellow solid.
Electrospray Mass Spec: 503.1 (M+H).sup.+
To a solution of 0.4 g (0.79 mmol) of the pyridinium salt in
THF:MeOH:H.sub.2 O (2:1:1, 3 mL total) was added 0.037 g (0.88
mmol) of lithium hydroxide and the reaction was heated to reflux
overnight. The reaction was neutralized with 6M HCl solution and
extracted three times with ethyl acetate. The organics were
combined, washed with brine, dried over MgSO.sub.4, and
concentrated in vacuo to provide 282 mg (86%) of
4-[(4-hydroxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-carboxyli
c acid methyl ester. Electrospray Mass Spec: 412.0 (M+H).sup.+
EXAMPLE 43
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-carb
oxylic acid hydroxyamide
According to the procedure of Example 5 242 mg (0.58 mmol) of the
product of Example 42 and 0.049 ml (0.65 mmol) of 2-butyn-1-ol
provides 0.239 g (98%) of
4-[(4-but-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-car
boxylic acid methyl ester. Electrospray Mass Spec: 464.2
(M+H)+.
According to the procedure of Example 38 0.239 g (0.58 mmol) of the
ester was hydrolyzed to provide 0.2 g (76%) of
4-[(4-but-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-car
boxylic acid. Electrospray Mass Spec: 448.3.0 (M-H)-
According to the procedure of Example 39 0.20 g (0.44 mmol) of the
carboxylic acid was converted to the hydroxamic acid
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-car
boxylic acid hydroxyamide providing 0.150 g (73%) of pure product.
Electrospray Mass Spec: 465.0 (M+H).sup.+
EXAMPLE 44
5-Bromo-3-methyl-2-{methyl-[4-(3-phenyl-prop-2-ynyloxy)-benzenesulfonyl]-am
ino}-benzoic acid
To a solution of 0.240 g (0.455 mmol) of the product of Example 11
in 6.0 mL of THF/methanol (1:1) was added 2.3mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was triturated with
ether/hexanes (1:1) and the solid was collected and dried in vacuo
to provide 0.181 g (77%) of the desired carboxylic acid product as
a white solid. Electrospray Mass Spec: 513.7 (M+H).sup.+
EXAMPLE 45
5-Bromo-2-({4-[3-(3-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid
To a solution of 0.251 g (0.450 mmol) of the product of Example 12
in 6.0 mL of THF/methanol (1:1) was added 2.3mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was triturated with
ether/hexanes (1:1) and the solid was collected and dried in vacuo
to provide 0.184 g (75%) of the desired carboxylic acid product as
a pale yellow solid. Electrospray Mass Spec: 543.8 (M+H).sup.+
EXAMPLE 46
5-Bromo-2-({4-[3-(2-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid
To a solution of 0.264 g (0.473 mmol) of the product of Example 13
in 6.0 mL of THF/methanol (1:1) was added 2.4 mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was triturated with
ether/hexanes (1:1) and the solid was collected and dried in vacuo
to provide 0.159 g (62%) of the desired carboxylic acid product as
a white solid. Electrospray Mass Spec: 543.8 (M+H).sup.+
EXAMPLE 47
5-Bromo-2-({4-[3-(4-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid
To a solution of 0.217 g (0.389 mmol) of the product of Example 14
in 6.0 mL of THF/methanol (1:1) was added 1.9mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo. The residue was triturated with
ether/hexanes (1:1) and the solid was collected and dried in vacuo
to provide 0.191 g (90%) of the desired carboxylic acid product as
a white solid. Electrospray Mass Spec: 543.8 (M+H).sup.+
EXAMPLE 48
2-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-iodo-3-methyl-benzoic
acid
To a solution of 0. 102 g (0.199 mmol) of the product of Example 17
in 6.0 mL of THF/methanol (1:1) was added 1.0 mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with ethyl acetate, washed with brine and the
combined organics were then dried over MgSO.sub.4, filtered and
concentrated in vacuo to provide 0.089 g (90%) of the desired
carboxylic acid product as a white solid. Electrospray Mass Spec:
499.8 (M+H).sup.+
EXAMPLE 49
2-[Benzyl-(4-but-2-ynyloxy-benzenesulfonyl)-amino]-3,5-dimethyl-benzoic
acid
To a solution of 0.161 g (0.338 mmol) of the product of Example 20
in 4.0 mL of THF/methanol (1:1) was added 1.7 mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and acidified with 5% HCl solution. The
mixture was extracted with dichloromethane and the combined
organics were then dried over MgSO.sub.4, filtered and concentrated
in vacuo to provide 0.151 g (97%) of the desired carboxylic acid
product as a white solid. Electrospray Mass Spec: 464.0
(M+H).sup.+
EXAMPLE 50
5-Bromo-3-metlhyl-2-{methyl-[4-(4-pyrrolidin-1-yl-but-2-ynyloxy)-benzenesul
fonyl]-amino}-benzoic acid
To a solution of 0.157 g (0.293 mmol) of the product of Example 23
in 4.0 mL of THF/methanol (1:1) was added 1.5 mL of 1.0N sodium
hydroxide solution. The reaction was heated to reflux overnight,
cooled to room temperature and brought to pH6-7 with 5% HCl
solution. The mixture was extracted with dichloromethane and the
combined organics were then dried over Na.sub.2 SO.sub.4, filtered
and concentrated in vacuo to provide 0.139 g (91%) of the desired
carboxylic acid product as a white solid. Electrospray Mass Spec:
520.9 (M+H).sup.+
EXAMPLE 51
5-Bromo-2-{[4-(4-diethylamino-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino}
-3-methyl-benzoic acid
According to the procedure of Example 50 0.180 g (0.335 mmol) of
the product of Example 24 provides 0.175 g (100%) of the desired
carboxylic acid as a tan foam. Electrospray Mass Spec: 522.9
(M+H).sup.+
EXAMPLE 52
5-Bromo-3-methyl-2-[methyl-(4-prop-2-ynyloxy-benzenesulfonyl)-amino]-benzoi
c acid
According to the procedure of Example 38 0.250 g (0.553 mmol) of
the product of Example 5 provides 0.237 g (98%) of the desired
carboxylic acid as a white solid. Electrospray Mass Spec: 435.8
(M-H).sup.-
EXAMPLE 53
5-Bromo-N-hydroxy-3-methyl-2-[methyl-(4-prop-2-ynyloxy-benzenesulfonyl)-ami
no]-benzamide
According to the procedure of Example 39 0.173 g of the product of
Example 52 provides 0.176 g (98%) of the hydroxamic acid as a white
solid. Electrospray Mass Spec: 452.8 (M+H).sup.+
EXAMPLE 54
5-Benzofuran-2-yl-2-[(4-methoxy-benzenesulfonyl)-pyridin-3-ylmethyl-amino]-
3-methyl-benzoic acid methyl ester
According to the procedure of Example 39 0.084 g of the product of
Example 6 provides 0.087 g (100%) of the hydroxamic acid as a white
foam. Electrospray Mass Spec: 466.8 (M+H).sup.+
EXAMPLE 55
5-Bromo-N-hydroxy-3-methyl-2-[methyl-(4-pent-2-ynyloxy-benzenesulfonyl)-ami
no]-benzamide
According to the procedure of Example 39 0.162 g of the product of
Example 7 provides 0.135 g (81%) of the hydroxamic acid as a tan
foam. Electrospray Mass Spec: 480.8 (M+H).sup.+Example 56
5-Bromo-2-[(4-hept-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-met
hyl-benzamide
According to the procedure of Example 39 0.180 g of the product of
Example 8 provides 0.127 g (69%) of the hydroxamic acid as a tan
foam. Electrospray Mass Spec: 508.8 (M+H).sup.+
EXAMPLE 57
5-Bromo-2-[(4-hex-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-meth
yl-benzamide
According to the procedure of Example 39 0.128 g of the product of
Example 10 provides 0.087 g (100%) of the hydroxamic acid as a
clear glass. Electrospray Mass Spec: 497.0 (M+H)+
EXAMPLE 58
5-Bromo-N-hydroxy-2-{[4-(4-methoxy-but-2-ynyloxy)-benzenesulfonyl]-methyl-a
mino}-3-methyl-benzamide
According to the procedure of Example 39 0.064 g of the product of
Example 26 provides 0.062 g (100%) of the hydroxamic acid as a tan
foam. Electrospray Mass Spec: 496.8 (M+H).sup.+
EXAMPLE 59
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(3-phenyl-prop-2-ynyloxy)-benzenesu
lfonyl]-amino}-benzamide
According to the procedure of Example 39 0.146 g of the product of
Example 44 provides 0.141 g (94%) of the hydroxamic acid as a pale
yellow foam. Electrospray Mass Spec: 528.8 (M+H).sup.+
EXAMPLE 60
5-Bromo-N-hydroxy-2-({4-[3-(3-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide
According to the procedure of Example 39 0.154 g of the product of
Example 45 provides 0.151 g (96%) of the hydroxamic acid as a light
orange solid. Electrospray Mass Spec: 558.8 (M+H).sup.+
EXAMPLE 61
5-Bromo-N-hydroxy-2-({4-[3-(2-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide
According to the procedure of Example 39 0.135 g of the product of
Example 46 provides 0.132 g (95%) of the hydroxamic acid as a white
foam. Electrospray Mass Spec: 558.9 (M+H).sup.+
EXAMPLE 62
5-Bromo-N-hydroxy-2-({4-[3-(4-methoxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide
According to the procedure of Example 39 0.158 g of the product of
Example 47 provides 0.116 g (72%) of the hydroxamic acid as a pale
yellow foam. Electrospray Mass Spec: 558.9 (M+H).sup.+
EXAMPLE 63
2-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-N-hydroxy-5-iodo-3-methy
l -benzamide
According to the procedure of Example 39 0.109 g of the product of
Example 48 provides 0.112 g (100%) of the hydroxamic acid as a
white foam. Electrospray Mass Spec: 514.8 (M+H).sup.+
EXAMPLE 64
2-[Benzyl-(4-but-2-ynyloxy-benzenesulfonyl)-amino]-N-hydroxy-3,5-dimethyl-b
enzamide
According to the procedure of Example 39 0.135 g of the product of
Example 49 provides 0.134 g (96%) of the hydroxamic acid as a white
solid. Electrospray Mass Spec: 479.0 (M+H).sup.+
EXAMPLE 65
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(4-pyrrolidin-1-yl-but-2-ynyloxy)-b
enzenesulfonyl]-amino}-benzamide
To a 0.degree. solution of 0.33 mL (0.650 mmol) of a 2.0M solution
of oxalyl chloride in dichloromethane, diluted with 2.7 mL of
dichloromethane, is added 0.050 mL (0.650 mmol) of DMF and the
reaction is stirred for 15 minutes at 0.degree.. A solution of
0.113 g (0.217 mmol) of the carboxylic acid product of Example 50,
dissolved in 1 mL of DMF, was added to the reaction and the
resulting mixture is stirred for 1 h at room temperature and then
poured into a 0.degree. mixture of 0.7 mL of water, 3.4 mL of THF
and 0.2 mL of a 50% aqueous solution of hydroxylamine. The reaction
is allowed to warm to room temperature overnight and the organics
are then concentrated in vacuo. The residue is diluted with ethyl
acetate, washed with water and saturated sodium bicarbonate, dried
over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo to
provide 0.087 g (75%) of the hydroxamic acid as a tan foam.
To a solution of 0.065 g (0.121 mmol) of the amino-hydroxamic acid
dissolved in 2.0 mL of dichloromethane was added 0.24 mL (0.24
mmol) of a 1.0M solution of HCl in ether. The resulting mixture was
stirred for 1 h at room temperature and then diluted with ether.
The precipitate was filtered, washed with ether and dried in vacuo
to provide 0.064 g (93%) of the hydrochloride salt of the
amino-hydroxamic acid as a brown solid. Electrospray Mass Spec:
535.9 (M+H).sup.+
EXAMPLE 66
5-Bromo-2-{[4-(4-diethylaniino-but-2-ynyloxy)-benzenesulfonyl]-methyl-amino
}-N-hydroxy-3-methyl-benzamide
To a 0.degree. solution of 0.38 mL (0.757 mmol) of a 2.0M solution
of oxalyl chloride in dichloromethane, diluted with 3.1 mL of
dichloromethane, is added 0.059 mL (0.757 mmol) of DMF and the
reaction is stirred for 15 minutes at 0.degree.. A solution of
0.132 g (0.252 mmol) of the carboxylic acid product of Example 51,
dissolved in 1 mL of DMF, was added to the reaction and the
resulting mixture is stirred for 1 h at room temperature and then
poured into a 0.degree. mixture of 0.8 mL of water, 3.9 mL of THF
and 0.24 mL of a 50% aqueous solution of hydroxylamine. The
reaction is allowed to warm to room temperature overnight and the
organics are then concentrated in vacuo. The residue is diluted
with ethyl acetate, washed with water and saturated sodium
bicarbonate, dried over Na.sub.2 SO.sub.4, filtered and
concentrated in vacuo to provide 0.126 g (93%) of the hydroxamic
acid as a tan foam.
To a solution of 0.093 g (0.173 mmol) of the amino-hydroxamic acid
dissolved in 3.0 mL of dichloromethane was added 0.35 mL (0.35
mmol) of a 1.0M solution of HCl in ether. The resulting mixture was
stirred for 1 h at room temperature and then diluted with ether.
The precipitate was filtered, washed with ether and dried in vacuo
to provide 0.091 g (92%) of the hydrochloride salt of the
amino-hydroxamic acid as a tan solid. Electrospray Mass Spec: 540.0
(M+H).sup.+
EXAMPLE 67
5-Bromo-2-[(4-but-2-ynyloxy-benzenesulfonyl)-(4-methyl-piperazin-1-ylmethyl
)-amino]-N-hydroxy-3-methyl-benzamide
According to the procedure of Example 50 0.128 g (0.228 mmol) of
the product of Example 22 provided 0.117 g (94%) of the carboxylic
acid. Electrospray Mass Spec: 550.0 (M+H).sup.+
To a 0.degree. solution of 0.27 mL (0.547 mmol) of a 2.0M solution
of oxalyl chloride in dichloromethane, diluted with 2.4 mL of
dichloromethane, is added 0.042 mL (0.547 mmol) of DMF and the
reaction is stirred for 15 minutes at 0.degree.. A solution of
0.100 g (0.182 mmol) of the carboxylic acid, dissolved in 1 mL of
DMF, was added to the reaction and the resulting mixture is stirred
for 1 h at room temperature and then poured into a 0.degree.
mixture of 0.5 mL of water, 2.7 mL of THF and 0.5 mL of a 50%
aqueous solution of hydroxylamine. The reaction is allowed to warm
to room temperature overnight and the organics are then
concentrated in vacuo. The residue is diluted with ethyl acetate,
washed with water and saturated sodium bicarbonate, dried over
Na.sub.2 SO.sub.4, filtered and concentrated in vacuo to provide
0.080 g (78%) of the hydroxamic acid as a tan foam. Electrospray
Mass Spec: 565.1 (M+H).sup.+
To a solution of 0.060 g (0.107 mmol) of the amino-hydroxamic acid
dissolved in 4.0 mL of dichloromethane was added 0.43 mL (0.43
mmol) of a 1.0M solution of HCl in ether. The resulting mixture was
stirred for 1 h at room temperature and then diluted with ether.
The precipitate was filtered, washed with ether and dried in vacuo
to provide 0.068 g (100%) of the bis-hydrochloride salt of the
piperazinehydroxamic acid as a tan solid. Electrospray Mass Spec:
565.0 (M+H).sup.+
EXAMPLE 68
5-Bromo-3-methyl-2-(methyl-{4-[4-(tetrahydro-pyran-2-yloxy)-but-2-ynyloxy]-
benzenesulfonyl}-amino)-benzoic acid
According to the procedure of Example 50 0.550 g (0.972 mmol) of
the product of Example 15 provided 0.448 g (84%) of the carboxylic
acid as a white solid. Electrospray Mass Spec: 549.9
(M-H).sup.-
EXAMPLE 69
5-Bromo-N-hydroxy-3-methyl-2-(methyl-{4-[4-(tetrahydro-pyran-2-yloxy)-but-2
-ynyloxy]-benzenesulfonyl}-amino)-benzamide
To a solution of 0.41 g (0.745 mmol) of the product of Example 68
in 4.0 mL of DMF was added 0.121 g (0.893 mmol) of 1-hydroxy
benzotriazole (HOBT) followed by 0.190 g (0.990 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).
The resultin mixture was stirred at room temperature for 1 h and
then 0.23 mL of a 50% aqueous solution of hydroxylamine was added
and the reaction was stirred overnight. The reaction was then
diluted with ethyl acetate and washed with water, 5% HCl solution
and saturated sodium bicarbonate solution. The organics were dried
over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo to
provide 0.337 g (80%) of the hydroxamic acid as a white foam.
Electrospray Mass Spec: 568.8 (M+H).sup.+
EXAMPLE 70
5-Bromo-N-hydroxy-2-{[4-(4-hydro-but-2-ynyloxy)-benzenesulfony]-methyl-amin
o}-3-methyl-benzamide
To a solution of 0.274 g (0.483 mmol) of the product of Example 69
in 4.0 mL of methanol was added 0.012 g (0.048 mmol) of pyridinium
p-toluenesulfonate and the resulting mixture was heated to reflux
for 18 h. The reaction was then concentrated in vacuo, diluted with
ethyl acetate and washed with 5% HCl solution, water and saturated
sodium bicarbonate solution. The organics were dried over Na.sub.2
SO.sub.4, filtered and concentrated in vacuo to provide 0.159 g
(68%) of the hydroxamic acid as a white powder. Electrospray Mass
Spec: 482.8 (M+H).sup.+
EXAMPLE 71
4-[(4-Hydroxy-benzenesulfonyl)-methyl-amino]-5-methyl-biphenyl-3-carboxylic
acid methyl ester
To 28 mL of degassed ethylene glycol dimethyl ether was added 2.15
g (5.19 mmol) of the product of Example 4, 0.696 g (5.71 mmol) of
phenylboronic acid, 0.300 g (0.26 mmol) of
tetrakis(triphenylphosphine)palladium(0), and 10.4 mL (20.8 mmol)
of 2M Na.sub.2 CO.sub.3 and the mixture was refluxed under nitrogen
for 18 h. The reaction was then cooled, diluted with ethyl acetate,
washed with water and brine, dried over Na.sub.2 SO.sub.4, filtered
and concentrated in vacuo. The residue was triturated with 1:3
dichloromethane/hexanes to provide 1.89 g (89%) of the desired
biphenyl product as a pale orange solid. Electrospray Mass Spec
412.4 (M+H).sup.+
EXAMPLE 72
4-{[4-(tert-Butyl-dimethyl-silanyloxy)-benzenesulfonyl]-methyl-amino}-5-met
hyl-biphenyl-3-carboxylic acid methyl ester
A mixture of 1.89 g (4.6 mmol) of the product of Example 71, 0.833
g (5.52 mmol) of t-butyldimethylsilyl chloride and 0.783 g (1.51
mmol) of imidazole in 8 mL of DMF was stirred at room temperature
for 18 h. The reaction was quenched with water and extracted with
dichloromethane. The organic layer was separated, washed with
brine, dried over Na.sub.2 SO.sub.4 and concentrated in vacuo. The
residue was chromatographed on silica gel eluting with ethyl
acetate/hexanes (1:20) to provide 1.89 g (78%) of the desired silyl
ether product as a white solid. Electrospray Mass Spec 526.0
(M+H).sup.+
EXAMPLE 73
4-[(4-Hydroxy-benzenesulfonyl)-methyl-amino]-5-(4-methyl-piperazin-1-ylmeth
yl)-biphenyl-3-carboxylic acid methyl ester
A mixture of 1.84 g (3.5 mmol) of the product of Example 72 and
0.748 g (4.2 mmol) of N-bromosuccinimide in 35 mL of carbon
tetrachloride was refluxed with sun lamp under nitrogen for 2.5 h.
The reaction was cooled, washed with water and brine, dried over
Na.sub.2 SO.sub.4 and concentrated in vacuo to give 2.33 g of the
benzylic bromide. The bromide was combined with 0.350 g (3.5 mmol)
of N-methyl piperazine and 1.45 g (10.5 mmol) of K.sub.2 CO.sub.3
in 20 mL of DMF and the mixture was stirred at room temperature for
18 h. The reaction mixture was diluted with dichloromethane, washed
with water and brine, dried over Na.sub.2 SO.sub.4 and concentrated
in vacuo. The residue was chromatographed on silica gel eluting
with 2% methanol/dichloromethane to provide 1.33 g (74%) of the
desired product as a pale yellow solid. Electrospray Mass Spec
510.0 (M+H).sup.+
EXAMPLE 74
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-(4-methyl-piperazin-1-
yl methyl)-biphenyl-3-carboxylic acid methyl ester
To a solution of 0.810 g (1.59 mmol) of the product of Example 73
and 0.594 mL (7.95 mmol) of 2-butyn-1-ol in 8 mL of THF, was added
2.08 g (7.95 mmol) of triphenylphosphine and then 1.25 mL (7.95
mmol) of diethyl azodicarboxylate. The mixture was stirred at room
temperature for 18 h, diluted with ethyl acetate, washed with water
and brine, dried over Na.sub.2 SO.sub.4, and concentrated in vacuo.
The residue was chromatographed on silica gel with 2%
methanol/dichloromethane to provide 0.830 g (93%) of the desired
product as a beige solid. Electrospray Mass Spec 562.1
(M+H).sup.+
EXAMPLE 75
4-[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-(4-methyl-piperazin-1-
yl methyl)-biphenyl-3-carboxylic acid
A mixture of 0.965 g (1.72 mmol) of the product of Example 74 and
8.6 mL (8.59 mmol) of 1N NaOH in 8.6 mL of THF and 8. 6 mL of
methanol was heated to reflux for 18 h. The reaction mixture was
cooled and neutralized with 3N HCl. The organic solvents were
removed and the resulting aqueous solution was extracted with
dichloromethane. The organic layer was separated, washed with water
and brine, dried over Na.sub.2 CO.sub.3 and concentrated in vacuo.
The residue was triturated with ether to provide 0.829 g (89%) of
the desired product as a cream solid. Electrospray Mass Spec 548.1
(M+H).sup.+
EXAMPLE 76
4-
[(4-But-2-ynyloxy-benzenesulfonyl)-methyl-amino]-5-(4-methyl-piperazin-1-y
lmethyl)-biphenyl-3-carboxylic acid hydroxyamide dihydro-chloride
salt
To a solution of 0.244 mL (0.487 mmol) of a 2M solution of oxalyl
chloride in dichloromethane at 0.degree. C. was added 0.038 mL
(0.487 mmol) of DMF and the mixture was stirred for 1 h at room
temperature. A solution of 0.089 g (0.163 mmol) of the product of
Example 75 in 0.5 mL dichloromethane was then added to the reaction
mixture and the resulting mixture was stirred for 1 h at room
temperature.
In a separate flask, a mixture of 0.149 mL (2.44 mmol) of 50%
aqueous hydroxylamine in 2.5 mL THF and 0.5 mL water was cooled for
15 min at 0.degree. C. and the acid chloride solution was added to
it in one portion. The resulting solution was allowed to warm to
room temperature with stirring overnight. The reaction mixture was
diluted with dichloromethane and washed with water and brine, dried
over NA.sub.2 SO.sub.4, filtered and concentrated in vacuo. The
residue was triturated with ether to provide 0.066 g (72%) of the
desired hydroxamic acid as a beige solid.
To a solution of 0.240 g (0.418 mmol) of the hydroxamic acid in 5
mL dichloromethane, was added 1.67 mL (1.67 mmol) of 1M HCl/ether
solution. The reaction was stirred for 1 h and then diluted with
ether. The resulting precipitate was filtered and dried in vacuo to
provide 0.245 g (92%) of the desired hydrochloride salt as a beige
solid. Electrospray Mass Spec 563.1 (M+H).sup.+
EXAMPLE 77
5-Bromo-3-methyl-2-{methyl-[4-(1-methyl-prop-2-ynyloxy)-benzenesulfonyl]-am
ino}-benzoic acid methyl ester
According to the procedure of Example 5, 0.400 g (0.966 mmol) of
the product of Example 4 and 0.083 mL (1.063 mmol) of 3-butyn-2-ol
provided 0.266 g (59%) of the propargylic ether as a colorless oil.
Electrospray Mass Spec 465.8 (M+H).sup.+
EXAMPLE 78
5-Bromo-3-methyl-2-{methyl-[4-(1-methyl-prop-2-ynyloxy)-benzenesulfonyl]-am
ino}-benzoic acid
According to the procedure of Example 10, 0.241 g of the product of
Example 77 was hydrolyzed with 2.6 mL of 1.0N sodium hydroxide
solution to provide 0.073 g (31%) of the desired carboxylic acid as
a white solid and 0.034 g (14%) of the starting ester. Electrospray
Mass Spec 451.8 (M+H).sup.+
EXAMPLE 79
5-Bromo-N-hydroxy-3-methyl-2-{methyl-[4-(1-methyl-prop-2-ynyloxy)-benzenesu
lfonyl]-amino}-benzamide
According to the procedure of Example 39, 0.068 g (0.150 mmol) of
the product of Example 34 provided 0.070 g (100%) of the hydroxamic
acid as a white foam. Electrospray Mass Spec 466.9 (M+H).sup.+
EXAMPLE 80
3-[3-(tert-Butyl-dimethyl-silanyloxy)-phenyl]-prop-2-yn-1-ol
To a solution of 2.0 g (9.091 mmol) of 3-iodophenol in 10 mL of DMF
was added 1.55 g (0.023 mol) of imidazole and 1.64 g (0.011 mol) of
t-butyldimethylsilyl chloride and the resulting mixture was stirred
at room temperature for 48 h. The reaction was then diluted with
ether and washed with water, dried over Na.sub.2 SO.sub.4, filtered
and concentrated in vacuo. The residue was used in the next step
without purification.
To a solution of 3.04 g (9.091 mmol) of the silylated iodoaryl in
55 mL of diethylamine was added 0.53 mL of propargyl alcohol
followed by 0.173 g (0.909 mmol) of copper (I) iodide and 0.32 g
(0.456 mmol) of bis(triphenyl-phosphine)palladium(II)dichloride.
The resulting mixture was stirred for 4 h at room temperature and
then concentrated in vacuo. The residue was chromatographed on
silica gel eluting with ethyl acetate/hexanes (1:10) to provide
0.73 g (31%) of the aryl acetylene as a brown oil. EI Mass Spec 262
(M.sup.+)
EXAMPLE 81
5-Bromo-2-({4-[3-(3-hydroxy-phenyl)-prop-2-ynyloxy]-benzenesulfonyl}-methyl
-amino)-3-methyl-benzoic acid
According to the procedure of Example 5, 0.158 g (0.604 mmol) of
the product of Example 80 and 0.250 g (0.604 mmol) of the product
of Example 4 provided 0.315 g of the propargylic ether.
According to the procedure of Example 10, 0.315 g of the
propargylic ether provided 0.169 g (67%) of the phenol-carboxylic
acid as a tan solid. Electrospray Mass Spec 527.9 (M-H).sup.-
EXAMPLE 82
5-Bromo-2-[(4-{3-[3-(tert-butyl-dimethyl-silanyloxy)-phenyl]-prop-2-ynyloxy
}-benzenesulfonyl)-methyl-amino]-3-methyl-benzoic acid
To a solution of 0.144 g (0.271 mmol) of the product of Example 81
in 1.0 mL of DMF was added 0.092 g (1.356 mmol) of imidazole and
0.098 g (0.651 mmol) of t-butyldimethylsilyl chloride and the
mixture was stirred at room temperature for 15 h. The reaction
mixture was then poured into 20 mL of water and stirred for 5 h and
then extracted with ether. The organics were washed with water,
dried over NA.sub.2 SO.sub.4, filtered and concentrated in vacuo.
The residue was chromatographed on silica gel eluting with ethyl
acetate/hexanes (1:3) to provide 0.136 g of the carboxylic
acidsilylated phenol as a white solid. Electrospray Mass Spec 643.8
(M+H).sup.+
EXAMPLE 83
5-Bromo-2-[(4-{3-[3-(tert-butyl-dimethyl-silanyloxy)-phenyl]-prop-2-ynyloxy
}-benzenesulfonyl)-methyl-amino]-N-hydroxy-3-methyl-benzamide
According to the procedure of Example 39, 0.097 g (0.150 mmol) of
the product of Example 82 provided 0.099 g of the hydroxamic acid
as a white solid. Electrospray Mass Spec 658.9 (M+H).sup.+
EXAMPLE 84
5-Bromo-N-hydroxy-2-({4-[3-(3-hydroxy-phenyl)-prop-2-ynyloxy]-benzenesulfon
yl}-methyl-amino)-3-methyl-benzamide
To a solution of 0.099 g (0.150 mmol) of the product of Example 83
in 1 mL of acetonitrile was added 3 mL of a 5% solution of 48% HF
in acetonitrile and the resulting solution was stirred at room
temperature for 15 h. The reaction mixture was then diluted with
water and ethyl acetate. The organic layer was washed with water,
dried over Na.sub.2 SO.sub.4 and concentrated in vacuo to give
0.031 g (38%) of the phenol hydroxamic acid as a pale yellow solid.
Electrospray Mass Spec 544.9 (M+H).sup.+
EXAMPLE 85
4-But-2-ynyloxy-benzenesulfonic acid sodium salt
To a solution of 52.35 g (0.225 mol) of 4-hydroxybenzenesulfonate
sodium salt in 1L of isopropanol and 225 mL of a 1.0N solution of
sodium hydroxide was added 59.96 g (0.45 mol) of 1-bromo-2-butyne.
The resulting mixture was heated to 70.degree. for 15 h and then
the isopropanol was removed by evaporation in vacuo. The resulting
white precipitate was collected by filtration, washed with
isopropanol and ether and dried in vacuo to give 56.0 g (100%) of
the butynyl ether as a white solid.
EXAMPLE 86
4-But-2-ynyloxy-benzenesulfonyl chloride
To a 0.degree. solution of 43.8 mL (0.087 mol) of 2M oxalyl
chloride/dichloromethane solution in 29 mL of dichloromethane was
dropwise added 6.77 mL (0.087 mol) of DMF followed by 7.24 g (0.029
mol) of the product of Example 85. The reaction mixture was stirred
for 10 minutes at 0.degree. then let warm to room temperature and
stirred for 2 days. The reaction was then poured into ice and
extracted with 150 mL of hexanes. The organics were washed with
water and brine, dried over Na.sub.2 SO.sub.4, filtered and
concentrated in vacuo to provide 6.23 g (88%) of the sulfonyl
chloride as a yellow solid; m.p. 63-65.degree. C. EI Mass Spec:
243.9 MH.sup.+
EXAMPLE 87
But-2-ynyloxy-benzene
According to the procedure of Example 5, 2.00 g (0.021 mol) of
phenol and 1.64 g (0.023 mol) of 2-butyn-1-ol provided 2.18 g (70%)
of the butynyl ether as a clear liquid. EI Mass Spec: 146.0
MH.sup.+
EXAMPLE 88
4-But-2-ynyloxy-benzenesulfonyl chloride
To a solution of 0.146 g (1.0 mmol) of the product of Example 87 in
0.3 mL of dichloromethanein an acetone/ice bath under N.sub.2 was
dropwise added a solution of 0.073 mL (1.1 mmol) of chlorosulfonic
acid in 0.3 mL of dichloromethane. After the addition was complete,
the ice bath was removed and the reaction was stirred at room
temperature for 2 h. To the reaction was then dropwise added 0.113
mL (1.3 mmol) of oxalyl chloride, followed by 0.015 mL DMF. The
reaction was heated to reflux for 2 h and then diluted with hexane
and poured into ice water. The organic layer was washed with brine,
dried over sodium sulfate, and concentrated in vacuo to provide
0.130 mg (53%) of the desired product as a light brown solid.
Pharmacology
Representative compounds of this invention were evaluated as
inhibitors of the enzymes MMP-1, MMP-9, MMP-13 and TNF-.alpha.
converting enzyme (TACE). The standard pharmacological test
procedures used, and results obtained which establish this
biological profile are shown below.
Test Procedures for Measuring MMP-1, MMP-9, and MMP-13
Inhibition
These standard pharmacological test procedures are based on the
cleavage of a thiopeptide substrates such as
Ac-Pro-Leu-Gly(2-mercapto-4-methyl-pentanoyl)-Leu-Gly-OEt by the
matrix metalloproteinases MMP-1, MMP-13 (collagenases) or MMP-9
(gelatinase), which results in the release of a substrate product
that reacts colorimetrically with DTNB
(5,5'-dithiobis(2-nitro-benzoic acid)). The enzyme activity is
measured by the rate of the color increase. The thiopeptide
substrate is made up fresh as a 20 mM stock in 100% DMSO and the
DTNB is dissolved in 100% DMSO as a 100 mM stock and stored in the
dark at room temperature. Both the substrate and DTNB are diluted
together to 1 mM with substrate buffer (50 mM HEPES pH 7.5, 5 mM
CaCl.sub.2) before use. The stock of enzyme is diluted with buffer
(50 mM HEPES, pH 7.5, 5 mM CaCl.sub.2, 0.02% Brij) to the desired
final concentration. The buffer, enzyme, vehicle or inhibitor, and
DTNB/substrate are added in this order to a 96 well plate (total
reaction volume of 200 .mu.l) and the increase in color is
monitored spectrophotometrically for 5 minutes at 405 nm on a plate
reader and the increase in color over time is plotted as a linear
line.
Alternatively, a fluorescent peptide substrate is used. In this
test procedure, the peptide substrate contains a fluorescent group
and a quenching group. Upon cleavage of the substrate by an MMP,
the fluorescence that is generated is quantitated on the
fluorescence plate reader. The assay is run in HCBC assay buffer
(50 mM HEPES, pH 7.0, 5 mM Ca.sup.+2, 0.02% Brij, 0.5% Cysteine),
with human recombinant MMP-1, MMP-9, or MMP-13. The substrate is
dissolved in methanol and stored frozen in 1 mM aliquots. For the
assay, substrate and enzymes are diluted in HCBC buffer to the
desired concentrations. Compounds are added to the 96 well plate
containing enzyme and the reaction is started by the addition of
substrate. The reaction is read (excitation 340 nm, emission 444
nm) for 10 min. and the increase in fluorescence over time is
plotted as a linear line.
For either the thiopeptide or fluorescent peptide test procedures,
the slope of the line is calculated and represents the reaction
rate. The linearity of the reaction rate is confirmed (r.sup.2
>0.85). The mean (x.+-.sem) of the control rate is calculated
and compared for statistical significance (p<0.05) with
drug-treated rates using Dunnett's multiple comparison test.
Dose-response relationships can be generated using multiple doses
of drug and IC.sub.50 values with 95% CI are estimated using linear
regression.
Test Procedure for Measuring TACE Inhibition
Using 96-well black microtiter plates, each well receives a
solution composed of 10 .mu.L TACE (final concentration 1
.mu.g/mL), 70 .mu.L Tris buffer, pH 7.4 containing 10% glycerol
(final concentration 10 mM), and 10 .mu.L of test compound solution
in DMSO (final concentration 1 .mu.M, DMSO concentration <1%)
and incubated for 10 minutes at room temperature. The reaction is
initiated by addition of a fluorescent peptidyl substrate (final
concentration 100 .mu.M) to each well and then shaking on a shaker
for 5 sec.
The reaction is read (excitation 340 nm, emission 420 nm) for 10
min. and the increase in fluorescence over time is plotted as a
linear line. The slope of the line is calculated and represents the
reaction rate.
The linearity of the reaction rate is confirmed (r.sup.2 >0.85).
The mean (x.+-.sem) of the control rate is calculated and compared
for statistical significance (p<0.05) with drug-treated rates
using Dunnett's multiple comparison test. Dose-response
relationships can be generate using multiple doses of drug and
IC.sub.50 values with 95% CI are estimated using linear
regression.
Human Monocvtic THP-1 Cell Differentiation Assay For Soluble
Proteins (THP-1 Soluble Protein Assay)
Mitogenic stimulation of THP- 1 cells cause differentiation into
macrophage like cells with concomitant secretion of tumor necrosis
factor (TNF-a) and TNF receptor (TNF-R p75/80 and TNF-R p55/60) and
Interleukin-8 (IL-8), among other proteins. In addition,
non-stimulated THP-1 cells shed both the p75/80 and the p55/60
receptors over time. The release of membrane bound TNF-.alpha. and
possibly TNF-R p75/80 and TNF-R p55/60, but not IL-8, is mediated
by an enzyme called TNF-a converting enzyme or TACE. This assay can
be used to demonstrate either an inhibitory or a stimulatory
compound effect on this TACE enzyme and any cytotoxic consequence
of such a compound.
THP-1 cells (from ATCC) are a human monocytic cell line which were
obtained from the peripheral blood of a one year old male with
acute monocytic leukemia. They can be grown in culture and
differentiated into macrophage like cells by stimulation with
mitogens.
For the assay, THP-1 cells are seeded from an ATCC stock which was
previously grown and frozen back at 5.times.106/ml/vial. One vial
is seeded into a T25-flask with 16 mls of RPMI-1640 with glutamax
(Gibco) media containing 10 % fetal bovine serum, 100 units/ml
penicillin, 100 .mu.g/ml streptomycin, and 5.times.10.sup.-5 M
2-mercapto-ethanol (THP-1 media). Each vial of cells are cultured
for about two weeks prior to being used for an assay and then are
used for only 4 to 6 weeks to screen compounds. Cells are
subcultured on Mondays and Thursdays to a concentration of
1.times.105/ml.
To perform an assay, the THP-1 cells are co-incubated in a 24 well
plate with 50 ml/well of a 24 mg/ml stock of Lipopolysacharide
(LPS) (Calbiochem Lot# B13189) at 37.degree. C. in 5% CO.sub.2 at a
concentration of 1.091.times.10.sup.6 cells/ml (1.1 ml/well) for a
total of 24 hours. At the same time, 50 ml/well of drug, vehicle or
THP-1 media is plated in appropriate wells to give a final volume
of 1.2 ml/well. Standard and test compounds are dissolved in DMSO
at a concentration of 36 mM and diluted from here to the
appropriate concentrations in THP-1 media and added to the wells at
the beginning of the incubation period to give final concentrations
of 100 mM, 30 mM, 10 mM, 3 mM, 1 mM, 300 nM, and 100 nM. Cell
exposure to DMSO was limited to 0.1 % final concentration. Positive
control wells were included in the experiment which had mitogen
added but no drug. Vehicle control wells were included as well,
which were identical to the positive control wells, except that
DMSO was added to give a final concentration of 0.083%. Negative
control wells were included in the experiment which had vehicle but
no mitogen or drug added to the cells. Compounds can be evaluated
for their effect on basal (non-stimulated) shedding of the
receptors by replacing the LPS with 50 ml/well of THP-1 media.
Plates are placed into an incubator set at 5% CO2 and at 37.degree.
C. After 4 hours of incubation, 300 ml/well of tissue culture
supernatant (TCS) is removed for use in an TNF-a ELISA. Following
24 hours of incubation, 700 ml/well of TCS is removed and used for
analysis in TNF-R p75/80, TNF-R p55/60 and IL-8 ELISAs.
In addition, at the 24 hours timepoint, and the cells for each
treatment group are collected by resuspension in 500 .mu.l/well of
THP-1 media and transferred into a FACS tube. Two ml/tube of a 0.5
mg/ml stock of propidium iodide (PI) (Boerhinger Mannheim cat. #
1348639) is added. The samples are run on a Becton Dickinson
FaxCaliber FLOW cytometry machine and the amount of dye taken up by
each cell is measured in the high red wavelength (FL3). Only cells
with compromised membranes (dead or dying) can take up PI. The
percent of live cells is calculated by the number of cells not
stained with PI, divided by the total number of cells in the
sample. The viability values calculated for the drug treated groups
were compared to the viability value calculated for the vehicle
treated mitogen stimulated group ("vehicle positive control") to
determine the "percent change from control". This "percent change
from control" value is an indicator of drug toxicity.
The quantity of soluble TNF-a, TNF-R p75/80 and TNF-R p55/60 and
IL-8 in the TCS of the THP-1 cell cultures are obtained with
commercially available ELISAs from R&D Systems, by
extrapolation from a standard curve generated with kit standards.
The number of cells that either take up or exclude PI are measured
by the FLOW cytometry machine and visualized by histograms using
commercially available Cytologic software for each treatment group
including all controls.
Biological variability in the magnitude of the response of THP-1
cell cultures requires that experiments be compared on the basis of
percent change from "vehicle positive control" for each drug
concentration. Percent change in each soluble protein evaluated
from the "vehicle positive control" was calculated for each
compound concentration with the following formula: ##EQU1##
For the soluble protein (TNF-a, p75/80, p55/60, IL-8) studies under
stimulated conditions, the mean pg/ml of duplicate wells were
determined and the results expressed as percent change from
"vehicle positive control". For the soluble protein (p75/80 and
p55/60 receptors) studies under non-stimulated conditions, the mean
pg/ml of duplicate wells were determined and the results expressed
as percent change from "vehicle positive control" utilizing the
following formula: ##EQU2##
IC50 values for each compound are calculated by non-linear
regression analysis using customized software utilizing the JUMP
statistical package.
For the cell viability studies, the viabilities (PI exclusion) of
pooled duplicate wells were determined and the results expressed as
% change from "vehicle positive control". The viability values
calculated for the compound treated groups were compared to the
viability value calculated for the "vehicle positive control" to
determine "percent change from control" as below. This value
"percent change from control" is an indicator of drug toxicity.
##EQU3##
References
Bjomberg, F., Lantz, M., Olsson, I., and Gullberg, U. Mechanisms
involved in the processing of the p55 and the p75 tumor necrosis
factor (TNF) receptors to soluble receptor forms. Lymphokine
Cytokine Res. 13:203-211, 1994.
Gatanaga, T., Hwang, C., Gatanaga, M., Cappuccini, F., Yamamoto,
R., and Granger, G. The regulation of TNF mRNA synthesis, membrane
expression, and release by PMA- and LPS-stimulated human monocytic
THP-1 cells in vitro. Cellular Immun. 138:1-10, 1991.
Tsuchiya, S., Yamabe, M., Yamagughi, Y., Kobayashi, Y., Konno, T.,
and Tada, K. Establishment and characterization of a human acute
monocytic leukemia cell line (THP-1). Int. J. Cancer. 26:1711-176,
1980.
Results of the above in-vitro matrix metalloproteinase inhibition,
TACE inhibition and THP standard pharmacological test procedures
are given in Table I below.
Table I. Inhibition in MMP, TACE and THP assays:
TABLE 1 % Inhib. IC.sub.50 (nM) or % Inhibition (.mu.M) @ 3 .mu.M
Example # MMP-1 MMP-9 MMP-13 TACE THP 31 19% (10) 301 724 44 6 32
26% (10) 643 255 58 15 33 32% (10) 1205 908 29 14 34 39% (10) 790
383 127 22 36 114 11 21 32 14 39 2488 21 68 67 0 43 4243 578 518
135 76 53 113 15 52 11 55 54 1616 304 154 16 84 55 1228 800 289 12
48 56 53% (10) 389 701 34 16 57 2364 232 358 47 11 58 7803 387 233
11 46 59 3815 857 321 66 20 60 1029 671 935 91 1 61 52% (10) 1507
1199 193 0 62 3602 901 588 104 15 63 901 840 275 16 87 64 1187 830
312 105 48 65 3352 602 537 148 12 66 21% (10) 2827 2377 346 5 67
1658 166 252 25 94 69 25% (10) 396 177 30 19 70 3203 477 83 6.8 88
76 1923 28 47 42 95 79 1534 455 433 122 9 84 113 694 189 34 0
Based on the results obtained in the standard pharmacological test
procedures described above, the compounds of this invention were
shown to be inhibitors of the enzymes MMP-1, MMP-9, MMP-13 and
TNF-a converting enzyme (TACE) and are therefore useful in the
treatment of disorders such as arthritis, tumor metastasis, tissue
ulceration, abnormal wound healing, periodontal disease, graft
rejection, insulin resistance, bone disease and HIV infection.
The compounds of this invention are also useful in treating or
inhibiting pathological changes mediated by matrix
metalloproteinases such as atherosclerosis, atherosclerotic plaque
formation, reduction of coronary thrombosis from atherosclerotic
plaque rupture, restenosis, MMP-mediated osteopenias, inflammatory
diseases of the central nervous system, skin aging, angiogenesis,
tumor metastasis, tumor growth, osteoarthritis, rheumatoid
arthritis, septic arthritis, corneal ulceration, proteinuria,
aneurysmal aortic disease, degenerative cartilage loss following
traumatic joint injury, demyelinating diseases of the nervous
system, cirrhosis of the liver, glomerular disease of the kidney,
premature rupture of fetal membranes, infammatory bowel disease,
age related macular degeneration, diabetic retinopathy,
proliferative vitreoretinopathy, retinopathy of prematurity, ocular
inflammation, keratoconus, Sjogren's syndrome, myopia, ocular
tumors, ocular angiogenesis/neovascularization and corneal graft
rejection.
Compounds of this invention may be administered neat or with a
pharmaceutical carrier to a patient in need thereof. The
pharmaceutical carrier may be solid or liquid.
Applicable solid carriers can include one or more substances which
may also act as flavoring agents, lubricants, solubilizers,
suspending agents, fillers, glidants, compression aids, binders or
tablet-disintegrating agents or an encapsulating material. In
powders, the carrier is a finely divided solid which is in
admixture with the finely divided active ingredient. In tablets,
the active ingredient is mixed with a carrier having the necessary
compression properties in suitable proportions and compacted in the
shape and size desired. The powders and tablets preferably contain
up to 99% of the active ingredient. Suitable solid carriers
include, for example, calcium phosphate, magnesium stearate, talc,
sugars, lactose, dextrin, starch, gelatin, cellulose, methyl
cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,
low melting waxes and ion exchange resins.
Liquid carriers may be used in preparing solutions, suspensions,
emulsions, syrups and elixirs. The active ingredient of this
invention can be dissolved or suspended in a pharmaceutically
acceptable liquid carrier such as water, an organic solvent, a
mixture of both or pharmaceutically acceptable oils or fat. The
liquid carrier can contain other suitable pharmaceutical additives
such a solubilizers, emulsifiers, buffers, preservatives,
sweeteners, flavoring agents, suspending agents, thickening agents,
colors, viscosity regulators, stabilizers or osmo-regulators.
Suitable examples of liquid carriers for oral and parenteral
administration include water (particularly containing additives as
above, e.g., cellulose derivatives, preferable sodium carboxymethyl
cellulose solution), alcohols (including monohydric alcohols and
polyhydric alcohols, e.g., glycols) and their derivatives, and oils
(e.g., fractionated coconut oil and arachis oil). For parenteral
administration the carrier can also be an oily ester such as ethyl
oleate and isopropyl myristate. Sterile liquid carriers are used in
sterile liquid form compositions for parenteral administration.
Liquid pharmaceutical compositions which are sterile solutions or
suspensions can be utilized by, for example, intramuscular,
intraperitoneal or subcutaneous injection. Sterile solutions can
also be administered intravenously. Oral administration may be
either liquid or solid composition form.
The compounds of this invention may be administered rectally in the
form of a conventional suppository. For administration by
intranasal or intrabronchial inhalation or insufflation, the
compounds of this invention may be formulated into an aqueous or
partially aqueous solution, which can then be utilized in the form
of an aerosol. The compounds of this invention may also be
administered transdermally through the use of a transdermal patch
containing the active compound and a carrier that is inert to the
active compound, is non-toxic to the skin, and allows delivery of
the agent for systemic absorption into the blood stream via the
skin. The carrier may take any number of forms such as creams and
ointments, pastes, gels, and occlusive devices. The creams and
ointments may be viscous liquid or semi-solid emulsions of either
the oil in water or water in oil type. Pastes comprised of
absorptive powders dispersed in petroleum or hydrophilic petroleum
containing the active ingredient may also be suitable. A variety of
occlusive devices may be used to release the active ingredient into
the blood stream such as a semipermeable membrane covering a
reservoir containing the active ingredient with or without a
carrier, or a matrix containing the active ingredient. Other
occlusive devices are known in the literature.
The dosage to be used in the treatment of a specific patient
suffering a MMP or TACE dependent condition must be subjectively
determined by the attending physician. The variables involved
include the severity of the dysfunction, and the size, age, and
response pattern of the patient. Treatment will generally be
initiated with small dosages less than the optimum dose of the
compound. Thereafter the dosage is increased until the optimum
effect under the circumstances is reached. Precise dosages for
oral, parenteral, nasal, or intrabronchial administration will be
determined by the administering physician based on experience with
the individual subject treated and standard medical principles.
Preferably the pharmaceutical composition is in unit dosage form,
e.g., as tablets or capsules. In such form, the composition is
sub-divided in unit dose containing appropriate quantities of the
active ingredient; the unit dosage form can be packaged
compositions, for example packed powders, vials, ampoules,
prefilled syringes or sachets containing liquids. The unit dosage
form can be, for example, a capsule or tablet itself, or it can be
the appropriate number of any such compositions in package
form.
* * * * *